The alveolate protozoan parasite Perkinsus olseni has a unique life cycle, including a mobile zoospore in an aerobic water column, vegetative trophozoite in the host tissues, and dormant hypnospore in an anaerobic environment such as decomposing host tissues or subsurface of the sediment. In this study, P. olseni trophozoites were induced from the zoospores in vitro using a Dulbecco Modified Eagle’s:Ham’s F-12 (DME/Ham’s F-12, 1:2) fortified with antibiotics and supplemented with 5% fetal bovine serum, 50 mM HEPES buffer, 3.5 mM sodium bicarbonate, and 200 mM L-glutamine. In the growth media, P. olseni zoospores developed into trophozoites and reproduced within two weeks at 25 °C room temperature. During two weeks of culture, the trophozoites increased their cell size from a few microns to 34.4 ± 14.1 μm in diameter. Numerous small-sized daughter cells of the trophozoites could be observed 4 and 6 days after incubation, suggesting that the doubling time of the trophozoites in the media can be 4 to 6 days. The hypnospore stage and subsequent zoosporulation could also be induced from the trophozoite stage developed in the growth media, confirming that the trophozoites are vital, although they were produced in vitro. The Dulbecco’s modified Eagle’s:Ham’s F-12 (DME/Ham’s F-12, 1:2) growth medium was considered a method of choice in the mass production of P. olseni trophozoites in vitro, as previously applied in in-vitro culture of Perkinsus spp.