All eyes were on Suzuki Factory rider Espargaro as he was fastest in qualyfing at the Catalunya race last time out while he also started last year's race in the Netherlands in pole position.
And to be fair to the Spaniard he did all he could as he was posted a 1:32.858, which made him the first rider to dip under the 1:33 mark at the track, but unfortunately for him Rossi went even faster with a 1:32.627.
It was just that kind of day for the veteran Italian, who is starting in pole for the first time this season, as his Yamaha team-mate and the in-form rider in the series, Jorge Lorenzo, was only eighth fastest.
Espargaro had to settle for second place in the end with reigning World Champion Marc Marquez third fastest with a 1:32.886.
On the second row of the grid you will have the second Repsol Honda bike of Dani Pedrosa, Pol Espargaro on the Monster Yamaha Tech 3 and Andrea Iannone from the Factory Ducati team.
Lorenzo will start next to Cal Crutchlow on the CWM LCR Honda with the second Suzuki of Maverick Vinales behind him.
Espargaro started in pole position in Catalunya 13 days ago and he breezed through Q1 at Assen, with Danilo Petrucci joining him. Pramac Racing rider Petrucci initially set the pace with a 1:33.503, but the Factory Suzuki of Espargaro upped the mark to 1:33.380 to book his place in the second session.
Although he couldn't make it two in a row, it's still a very good follow-up from the Spaniard.
1 Valentino ROSSI Movistar Yamaha 1'32.627
2 Aleix ESPARGARO Team SUZUKI ECSTAR 0.231
3 Marc MARQUEZ Repsol Honda Team 0.259
4 Dani PEDROSA Repsol Honda Team 0.360
5 Pol ESPARGARO Monster Yamaha Tech 3 0.386
6 Andrea IANNONE Ducati Team 0.389
7 Cal CRUTCHLOW CWM LCR Honda 0.401
8 Jorge LORENZO Movistar Yamaha 0.415
9 Maverick VIÑALES Team SUZUKI ECSTAR 0.449
10 Andrea DOVIZIOSO Ducati Team 0.485
11 Danilo PETRUCCI Octo Pramac Racing 0.499
12 Bradley SMITH Monster Yamaha Tech 3 0.609
13 Stefan BRADL Athinà Forward Racing 0.409
14 Yonny HERNANDEZ Octo Pramac Racing 0.495
15 Loris BAZ Athinà Forward Racing 0.619
16 Scott REDDING EG 0,0 Marc VDS 0.691
17 Hector BARBERA Avintia Racing 0.843
18 Mike DI MEGLIO Avintia Racing 0.909
19 Eugene LAVERTY Aspar MotoGP Team 1.048
20 Jack MILLER CWM LCR Honda 1.265
21 Alvaro BAUTISTA Aprilia Racing Team Gresini 1.356
22 Nicky HAYDEN Aspar MotoGP Team 1.592
23 Alex DE ANGELIS E-Motion IodaRacing Team 1.809
24 Marco MELANDRI Aprilia Racing Team Gresini 2.996