Hypo-osmotic swelling test (HOST) is a method of assessing the functional integrity of sperm plasma membrane which in turn was claimed as indicator of sperm fertility. Standard protocol of HOST has been established indifferent species but not in buffaloes. Thus, the current study was conducted to establish the HOST assay for buffalo frozen-thawed sperm cells. Frozen-thawed sperm cells were incubated indifferent osmolalities (0, 50,100, 150, 200, 250, and 300 mOsm) of sodium citrate-fructose solution to assess sperm reaction. Then, the effectiveness of sucrose vs. fructose was compared as sugar component of the HOST solution. Results showed higher(P<0.05)number of HOS positive (+) spermatozoa in 150 mOsm compared to 0, 250 and 300 mOsm/L though no significant difference was observed with 50, 100, and 200 mOsm/L. Sucrose-and fructose-containing solutions are both effective in enhancing swelling among treated spermatozoa. The results demonstrated that 150 mOsm is the efficient osmolality to effect optimum reaction of frozen-thawed buffalo spermatozoaand that either sucrose or fructose could be used for HOST solution to assess the functional membrane integrity of buffalo sperm cells.