Background: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which evaluates cellular mitochondrial activity is widely used for the assessment of cell proliferation and viability. Objective: This study was performed to assess human sperm viability using MTT assay. Materials and Methods: In this laboratory study, human-ejaculated semen samples (n = 56 from different donors) were used. The sperm viability was determined using quantitative MTT assay and the sperm motility was assessed according to World Health Organization guidelines. Sperm viability and the correlation between sperm viability and motility were analyzed. Results: Data revealed a marked positive correlation between MTT reduction rate and the percentage of viable spermatozoa. The Pearson’s correlation coefficients also showed a significant correlation between sperm viability and motility. Conclusion: MTT assay which is based on mitochondrial functionality is a reliable method for evaluating human sperm viability and could be used as a diagnostic test for predicting sperm fertilization ability in clinical settings.