Cosby's lawyers had argued that Montgomery County prosecutors violated his rights by refusing to call his accuser, Andrea Constand, as a witness at his preliminary hearing in May 2016.
The decision to introduce her allegations through police detectives who took her statement in 2005 robbed Cosby of his right to cross-examine Constand, his attorneys said.
Bill Cosby charged with sexual assault: prosecutor
But the judge who will preside over the trial, Steven O'Neill, ruled the case could move forward, a decision that was upheld by a mid-level appeals court. The Pennsylvania Supreme Court on Wednesday declined to hear Cosby's last-ditch appeal.
The case is the only criminal prosecution to result from accusations of sexual assault brought against Cosby by more than 50 women, many of which allegedly occurred decades ago. Cosby has denied any wrongdoing.
Constand has accused Cosby of drugging and then sexually assaulting her in 2004 at his home in a Philadelphia suburb.
Two more women accuse Bill Cosby of sexual assault
Jury selection in the case is scheduled to begin on May 22, with the trial set for June 5.
Cosby's lawyers have unsuccessfully sought to have the charges dismissed several times since he was charged in December 2015.
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