The spinner was named in Pakistan's squad for Saturday's series-opener in Hambantota, but will instead be on his way to Australia to have his bowling action tested by the International Cricket Council (ICC).
Ajmal was reported during the sides' recent Test series, won 2-0 by Sri Lanka, and faces a potential ban if his action is found to be illegal.
Having taken 182 wickets in the 50-over format since making his debut in 2008, Ajmal will be a sore loss and Misbah has called on his other bowlers to step up and help them to an early lead.
Pakistan's squad shows a number of changes from the Test side as their ODI specialists, including Shaid Afridi and Mohammed Hafeez, arrive in Sri Lanka in their build-up for the World Cup next year.
"It will be a big miss," the captain said of Ajmal. "His exclusion is crucial but at the same time it will be a chance for others to stand up and fulfil what normally Ajmal provides us.
"Everyone is capable and they have to put some effort so that we don't feel his absence.
"We have got a specialist one-day side and that will mentally boost us in the field.
"Most of the players have adapted themselves according to the format and they know exactly what is required to shift their focus from Test to ODI.
"There are a lot of changes in the ODI squad and our specialist players are in really good touch and they know their role and understand what to do."
A lack of match action could hamper Pakistan given they have not played a 50-over match since March, whereas their hosts have played 10 times in the format across that spell.
There have been five victories for Sri Lanka in that time, including a series triumph in England, and they come into the match on the back of three-successive wins over Misbah's men.
Captain Angelo Mathews has similar problems to his opposing number, though, with off-spinner Sachithra Senanayake currently suspended for an illegal bowling action.
The 29-year-old has undergone remodelling on his delivery style but remains banned by the ICC until further tests are carried out.
Mathews will hope Rangana Herath can fill the void left by Senanayake after he took 23 wickets in the Test series, including nine in the first innings of the second match in Colombo.
Pace bowler Lasith Malinga will undergo a fitness test ahead of Saturday's game for Sri Lanka, while Junaid Khan remains a concern for the tourists due to concussion.