Spies went round the track at Assen in a time of one minute 34.866 seconds, with Crutchlow a runner-up in the second practice session this afternoon.
The Monster Tech 3 Yamaha rider continues to impress 11 days on from breaking and dislocating his left ankle during practice for his home race at Silverstone and was just 0.006 seconds behind Spies on Thursday, with Ducati's Nicky Hayden in third place.
Crutchlow, who was only ninth quickest this morning, put off an operation on his injured ankle to continue his MotoGP world championship challenge this week after courageously finishing sixth from the back of the grid at Silverstone.
"I am happy to be second quickest considering my physical condition is certainly still not the best after the crash at Silverstone," he said.
"There was quite a bit of pain with the ankle this morning and it took me longer than normal to get into a rhythm. But overall I am really pleased to be second because I am still quite sore.
"I am sure it is going to get better the more time I spend on the bike but I had arm pump this morning too because I was compensating for the ankle. But this afternoon it wasn't an issue at all."
Series leader Jorge Lorenzo, who won at Silverstone to extend his points lead to 25 over defending champion Casey Stoner, finished fifth this afternoon after topping the time sheets this morning.
Pedrosa - third in the overall standings - and Stoner were second and third this morning but also suffered this afternoon.
Repsol Honda rider Pedrosa finished sixth while team-mate Stoner was 10th.