The Samsung Crescent Racing Suzuki rider showed his speed in the final three minutes of the session to pull ahead of title rival Tommy Hill on the timesheets.
Michael Laverty (Swan Yamaha) was third and was followed by Jon Kirkham (Samsung Crescent Racing Suzuki) and Shane Byrne (HM Plant Honda)
"We had a definite advantage going into this weekend having done the WSB round earlier in the season," said Hopkins.
"I tried to keep the laps to a minimum today as the pain can be extreme when you don't have the adrenaline pumping as much as you would in qualifying or in a race.
"It has been a really tough day but I have my physio here and when I am not on the bike I am having therapy so that I can be the strongest I can for the races on Sunday."
BSB FP2 times
1. John Hopkins (Samsung Crescent Racing Suzuki) 2m:06.152s
2. Tommy Hill (Swan Yamaha) +0.559s
3. Michael Laverty (Swan Yamaha) +0.744s
4. Jon Kirkham (Samsung Crescent Racing Suzuki) +1.523s
5. Shane Byrne (HM Plant Honda) +1.780s
6. Peter Hickman (Tyco Honda) +1.804s
7. Graeme Gowland (WFR Honda) +1.869s
8. Michael Rutter (Rapid Solicitors-Bathams Ducati) +2.133s
9. Alex Lowes (Motorpoint Yamaha) +2.155s
10. Josh Brookes (Relentless Suzuki by TAS) +2.166s