The in vitro cultivation of Leishmania and Trypanosoma parasites plays an important role in the diagnosis and treatment of parasitic diseases. Although Evans’s modified Tobie and Novy–MacNeal–Nicolle media, for Leishmania spp. and Trypanosoma cruzi, respectively, are the two commonly used media for both isolation and maintenance of strains in vitro, their preparation is expensive and laborious and requires fresh rabbit blood from housed animals. The purpose of this study was to evaluate the in vitro growth of both parasites with an alternative monophasic, blood-free, easy, and affordable medium called RPMI-PY, which was previously demonstrated suitable for the in vitro growth of Leishmania infantum. The potential growth of different Leishmania species and Trypanosoma cruzi was evaluated in traditional culture media versus RPMI-PY medium, and we recorded the protozoa parasites’ morphology via orange acridine–ethidium bromide staining. The results of our study show that RPMI-PY medium can be used for Trypanosoma cruzi, Leishmania amazonensis, Leishmania major, and Leishmania tropica species since in all the species except Leishmania braziliensis, the exponential growth of the parasite was observed, in many cases higher than conventional media. The staining confirmed not only their growth during the 72 h investigation but also the optimal morphology and viability of the protozoa in the RPMI-PY medium.