The present study aimed to determine the impact of sperm capacitation on sperm rheotaxis and sperm kinematics within microfluidics of buffalo cryopreserved semen. semen straws (n = 24) were obtained from 3 fertile buffalo bulls. Each straw was divided into three aliquots: control aliquot, caffeine aliquot and heparin aliquot. Sperm rheotaxis and all sperm kinematics were determined through a computer-assisted sperm analysis (CASA) system using the microfluidic platform with controlled flow velocity. The results showed that the positive rheotaxis % (PR%) of caffeine and heparin were significantly higher (PR% = 49.4±1 and 48.8±1, respectively) than control (45.1±1) (p<0.0001). Sperm kinematics show a significant variation among the three aliquots. Results show no significant differences in VCL of caffeine and heparin compared to control. While VCL of caffeine aliquot was significantly higher than that of heparin (p=0.01). The VAP of the caffeine aliquot was significantly higher than that of heparin and control (p=0.05). VSL and BCF of caffeine and heparin aliquots were significantly higher than control (p<0.001 and p<0.0001, respectively). In conclusion, this study confirms that PR% and most sperm kinematics in buffalo frozen semen improved during invitro capacitation using caffeine and heparin.