The Saints and the Addicks, who have slipped down the ladder from the Premier League into League One, will meet at St Mary's in the pick of the Southern ties.
Former winners MK Dons are at home to Northampton, Swindon play host to Norwich and Leyton Orient are up against Hereford in the other matches.
In the Northern Section, Leeds will have their sights set on a quarter-final place having been handed a home draw with Grimsby Town.
Bury's reward for their shock victory over Tranmere, which cost John Barnes his job, is a trip to either Accrington or Shrewsbury, who saw their midweek meeting postponed due to a waterlogged pitch.
Bradford play host to Port Vale and Chesterfield welcome Carlisle to Saltergate in the remaining Northern fixtures.
Johnstone's Paint Trophy quarter-finals:
Northern Section: Leeds v Grimsby; Accrington/Shrewsbury v Bury; Bradford v Port Vale; Chesterfield v Carlisle.
Southern Section: Milton Keynes Dons v Northampton; Leyton Orient v Hereford; Swindon v Norwich; Southampton v Charlton.
Ties to be played in the week commencing 9th November 2009