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Itumeleng Khune, Eric Mathoho, Reneilwe Letsholonyane, Siphiwe Tshabalala, George Lebese and Bernard Parker are all doubtful starters for the encounter after forming part of the Bafana Bafana squad that played Poland in Warsaw last week and Kenya in Nairobi on Tuesday.
Kingston Nkatha and Lincoln Zvasiya were also on national duty - for Zimbabwe - in 2013 Africa Cup of Nations qualifiers.
Khune is almost certain to miss the match, according to Khumalo, who lashed out at the Premier Soccer League for not scheduling a later cup fixture, as the goalkeeper is nursing an ankle niggle and is also dealing with a family bereavement.
"It's too late. We can never change it," Khumalo said ahead of the match.
"We've got a team that will be able to represent the Kaizer Chiefs family and we will go out there and fight."
The home match for the Students will be played at Bidvest Stadium on the university campus in Johannesburg, rather than the larger Mbombela Stadium in Nelspruit.
Wits last played Chiefs in a league clash in Nelspruit, with Amakhosi running out 3-1 victors.
Khumalo compared the pitch at the smaller stadium to Chiefs' training ground.
"Definitely for them (Wits), they want a compact field, but that won't pose a problem for us because we also have a smaller pitch at the Village (in Naturena) where we do our training," he said.
Khumalo believed Wits would be a difficult prospect at home.
"When you look at their past two games - and the game we have played - definitely they might have been hurt," Khumalo said.
"The reason why probably they are taking the game to Bidvest is it will suit their style.
"We expect them to be very, very compact in terms of the width. They are working on their defence.
"I'm not going to single out an individual. Wits is a different team at home and they have marvellous players.
"They are very good in terms of going forward."
On Saturday, Ajax Cape Town host a strong SuperSport United side at Athlone Stadium and Golden Arrows travel to Bethlehem to take on Free State Stars.
Black Leopards are at home to defending champions Orlando Pirates in Polokwane and Moroka Swallows, recently crowned MTN8 champions, will hope to continue their cup form against Maritzburg United at Harry Gwala Stadium.
On Sunday, Chippa United face Bloemfontein Celtic in Cape Town and AmaZulu entertain Platinum Stars in Durban.
In Tuesday's final first-round fixture, league strugglers Mamelodi Sundowns will hope to get their season back on track when they host University of Pretoria in a derby clash at Loftus Versfeld.
