Both players were unused substitutes in Tuesdays defeat at Northampton, with Mullins missing out on a starting place due to a precaution on the ankle injury he picked up against Burton.
Ainsworth could be drafted in for Michael O'Connor who has yet to complete a full 90 minutes as he works his way back to match fitness after a pre-season injury.
Boss Steve Evans expects a tight game as the Millers continue their tough looking start.
He told BBC Radio Sheffield: "I don't deal with defeat well and I was up watching the DVD of the Northampton game at 4am on Wednesday morning. I think in our opening 10 games we play every one of the teams that I expect to be challenging for this division.
"Chesterfield are a good side, a passing side and they have a very good management team - we are expecting a tough, competitive derby."