Previous studies conducted in Leishmania led us to test the hypothesis that addition of human urine (HU) to the Liver Infusion Tryptose (LIT) medium would stimulate the in vitro growth of Trypanosoma cruzi and Trypanosoma rangeli strains. Herein, we show that the addition of 3% HU to LIT medium (LIT-HU3) significantly stimulated the growth of all the T. rangeli strains studied when compared with the parasite growth in conventional LIT medium (p<0.05), and it was equivalent to the growth observed in LIT supplemented with fetal calf serum (FCS) in two parasite strains. Four out of the six T. cruzi strains analyzed showed a significant increase in parasite multiplication in LIT-HU3 (p<0.05). However, two parasite strains presented good growth in both LIT and LIT-HU, suggesting differences in the parasite's ability to grow in vitro. Furthermore, we have not observed differences in T. cruzi growth in LIT-HU3 and LIT supplemented with heat-denatured HU and in the metacyclogenesis of parasite strains cultured in LIT-HU3. These results allow concluding that the addition of HU to LIT medium stimulates the in vitro growth of T. rangeli and T. cruzi and can replace FCS as a supplement in culture medium.