Chesterfield will host the Premiership outfit next month but it was Tranmere who ended the day in upbeat mood after an own goal and excellent finish from Gareth Taylor wrapped up the points.
It was an accomplished performance from the visitors who repeated last season's scoreline on this ground and deserved to win.
Chesterfield faded after a promising opening although it could have been a different story but for two crucial saves by Gavin Ward.
The imposing keeper made a fingertip interception to turn away a cross by Paul Shaw and then plunged to his left to save the striker's firm drive after a mistake by Ian Goodison had let him in.
After that Tranmere began to assert themselves in midfield and the first real threat to the home defence came when Chris Shuker found space at the back post in the 29th minute to volley straight at Barry Roche.
Shuker then delivered one of his trademark crosses but Taylor headed wide from 12 yards. When the goal eventually came it was virtually impossible to save.
Kevin Ellison had been harshly booked for diving but he made amends with a fierce right-foot shot from the edge of the box that Roche could only push out to John Mullin. He stabbed the rebound goalwards and Aaron Downes headed it into the roof of his own net as he desperately tried to clear.
The second half belonged to the visitors who became increasingly dominant in possession and always looked solid at the back.
Goodison and the impressive Chris McCready hardly put a foot wrong as once Tranmere scored a second the match was over.
The goal came in the 67th minute and sprang from a poor pass by Mark Allott.
Paul McLaren picked up the loose ball and sent a 40-yard pass through to Taylor who slipped between the two centre-halves before lobbing over Roche from the edge of the area.
Chesterfield always kept going but they looked tired after Wednesday night's extra-time exertions in the Carling Cup against Wolves and Tranmere comfortably ran the clock down to take the three points home.