Several laboratory methods to evaluate ram sperm quality have been developed. The combination of different fluorescent probes is suitable to simultaneously evaluate different sperm characteristics but the need for a fluorescent microscope restricts its use. The aim of the present study was to evaluate the efficacy of Hypoosmotic Trypan Blue Giemsa (HTBG) staining to simultaneously detect sperm morphological abnormalities, plasma and acrosomal membrane integrity using phase contrast and fluorescence microscopy as the gold standards. Samples from twelve fresh ejaculates from three rams (4 ejaculates/ram) were used in the study. Sperm cells were evaluated using HTBG, phase contrast (PC) and fluorescent (FLUO) techniques. HTBG was more effective in detecting sperm defects when compared to PC (P < 0.05). No significant differences (P > 0.05) were observed between HTBG and FLUO in the assessment of plasmatic membrane and acrosome integrity. High correlation between HTBG and FLUO techniques was observed when assessing plasma membrane and acrosome (R = 0.97 and 0.96, respectively). In conclusion, the HTBG staining is suitable to assess ram sperm morphology, plasma and acrossomal membrane integrity simultaneously.