Fetal Bovine Serum (FBS) is an expensive source of macronutrients which are required for proper nutrition of Leishmania parasite in the culture medium. An alternative, cost effective source of macronutrients which can replace the use of FBS in tissue culture medium is required. The potential of Soy Protein Isolate (SPI) to replace FBS in RPMI-1640 medium for the in vitro cultivation of Leishmania donovani was evaluated. Commercially available SPI powder was used in RPMI-1640 medium as a substitute of FBS to cultivate L. donovani promastigotes. The growth, multiplication and morphology of cultivated parasites was observed in conventional RPMI-1640 with 10% FBS (v/v) and RPMI-1640 containing 10% SPI (v/v) by using light microscopy, measurement of absorbance and cell counting. The growth of Leishmania promastigotes in the medium containing 10% SPI was slower in initial phase; however, the parasites were morphologically larger as compared to those in RPMI-1640 medium containing 10% FBS. Cell count in the SPI-containing RPMI-1640 medium was 2.3 × 108 cells/ml whereas it was 1.9 × 107 cells/ml in RPMI-1640 with 10% FBS. This study concludes that RPMI-1640 may be supplemented with SPI instead of FBS for the in vitro cultivation of Leishmania donovani promastigotes to decrease the culture maintenance cost in developing countries.