Polycarpa mytiligera is the only molecularly characterized solitary ascidian capable of regenerating any organ in the body. The cellular basis for regeneration in P. mytiligera is largely unknown, and methods for isolating live cells from this species for functional analyses are unavailable. Here, we developed a method for isolating live cells from P. mytiligera, overcoming major experimental challenges, including its dissociation of the thick body wall and native cellular autofluorescence. The ability to purify live cells will promote future studies of cell function in P. mytiligera regeneration.