The 32-year-old underwent a scan on the calf strain he suffered during Friday's 0-0 draw in Sweden on Sunday morning and the results have confirmed that he will be unable to play on Tuesday evening.
Wolves frontman Kevin Doyle, who was informed by text that he had not made the final 23 for the double-header, has been drafted in as a replacement.
A spokesman for the Football Association of Ireland said: "The Football Association of Ireland today [Sunday] confirmed that Robbie Keane has been ruled out of Tuesday's FIFA World Cup qualifier against Austria in Aviva Stadium.
"A scan this morning [Sunday] confirmed that Robbie is suffering from calf strain after Friday's game against Sweden. Wolves striker Kevin Doyle has been called up in place of Keane.
"The manager has spoken to Kevin, and he will link up with the squad in time for training in Malahide tomorrow [Monday] morning."
Trapattoni had earlier revealed that Keane has gone for a scan, but at the time, was hopeful the Los Angeles Galaxy striker, who was in line to win his 124th cap against the Austrians, would make the game.
The 74-year-old Italian now faces something of a selection headache with Jon Walters also struggling with a back injury and Doyle still coming to terms with his initial disappointment.
Trapattoni has been impressed with Derby's Conor Sammon and has handed Simon Cox key roles in the past, while Wes Hoolahan has impressed in a withdrawn role since being drafted into the squad.
However, the manager he has built his team around 54-goal Keane ever since his arrival and his absence leaves a sizeable hole.
The deserved point with which Trapattoni's men returned from Stockholm kept them firmly in the race for second place in Group C, although they dropped to fourth place as a result of Austria's 6-0 demolition of minnows the Faroe Islands.
However, with the Swedes not in action on Tuesday evening, victory over the Austrians would leave the Republic in possession, for the time being at least, of the runners-up spot, and no one within the camp is underestimating the importance of the fixture.
Asked if he would field the same team at the Aviva Stadium as he did at the Friends Arena, Trapattoni said, somewhat coyly: "Ninety-nine per cent, maybe.
But this game, we must think about all our possibilities. It's a game we want to win, but also those who don't start, the one or two or three changes [substitutions] will be very, very important."
Trapattoni delivered something of a curve ball in the run-up to the Sweden game when he initially named winger Robbie Brady in his team, and then revealed he was not certain he would in fact start, an attempt, he said, to spark a reaction from the Hull midfielder.
In the event, Trapattoni opted for a double change when Glenn Whelan's ankle injury prevented him from taking part, replacing the Stoke man with the more creative James McCarthy and asking Walters to patrol the right side of midfield instead.
Having already placed himself in the firing line by informing Doyle he would not be in the squad only by text, Trapattoni courted further criticism with his treatment of Brady, but was swift to attempt to clarify his decision on Sunday.
He said: "Reporters in the world are good at writing pieces, but I never said he was confused, what I wanted to say was that I wanted to stimulate him psychologically, I want to see him stronger psychologically.
"But I never said - or I didn't mean to say - that he was confused. The headlines then are a different story.
"There is no problem with Robbie. The player is ready, there is no problem. I know.
"There are situations when a manager wants to stimulate players. I said I wanted to see on the pitch what his condition was. He is not confused."
There was at least some positive injury news for Trapattoni on Sunday when Whelan returned to training, and he is expected to resume against Austria.
The manager went into the game in Stockholm knowing defeat might have signalled an end to his reign amid speculation that former Reading boss Brian McDermott was already being lined up as his replacement, but emerged from it, as he has done so often during his time at the helm to date, having been largely vindicated.
His decision to prefer 33-year-old David Forde to the action-starved Keiren Westwood paid off, as did the selections of Seamus Coleman, Ciaran Clark and Marc Wilson in a new-look defence and Paul Green, James McClean, McCarthy and Walters in midfield.
Trapattoni said: "Our young players, if they are here, it's because I trust them and I have confidence in them in the first place.
"We need to give them a chance to improve in every game. Every game improves their personality."