Hepatitis B surface antigen (HBsAg) is one of the most important serological markers used to diagnose acute and chronic hepatitis B infection. The objective of the current evaluation was to assess the operational characteristics of the Kenya Medical Research Institute (KEMRI) Hep-cell II against an ELISA Exsym HBsAg in the detection of hepatitis B surface antigens. To evaluate the Hep-cell II test, blood samples were collected from blood donors and processed for detection of HBsAg using Hep-cell II based on the test principle and procedure outlined by the manufacturer. ELISA Axsym HBsAg test was used as golden standard. Of the 400 samples tested, 287 (71.8%) were positive by Hep-cell test and 295 (73.8%) were positive by the ELISAAxsym. Hep-cell test had a sensitivity of 98.6% and specificity of 95.96%. Similar values of sensitivity and specificity of the Hep-cell test were obtained even when Bayesian Analysis Model was applied. The positive and negative predictive values of Hep-cell test were 98.61% and 95.96%, respectively. The positive and negative diagnostic likelihood ratios of Hep-cell test were 24.4% and 0.0145, respectively. In conclusion, the Hep-cell test is useful for detecting hepatitis B virus and the high likelihood ratio observed suggests that it may be useful in blood screening. However, it may be necessary to evaluate for cost-effectiveness and robustness in field conditions before the test is recommended for use.