Poliomyelitis, an acute viral infectious disease caused by poliovirus, still remains a public health problem in developing countries. Despite the global effort to eradicate polio, continuing the polio immunization with a potent and safe vaccine is essential. For accurate vaccine evaluation, three types of cell lines including Hela, Hep2C and Vero were evaluated and compared using two methods of polio vaccine potency tests (micro & macro). For cells comparison, five different batches from polio vaccines were tested and to develop the test, five variables including viruses, cells, serum, media and Co2 were studied. For validation, the titer of which has been well established as a working reference preparation (WRP) was applied to control the accuracy and reproducibility of the testing system. Multiple comparisons were performed by analysis of variance (ANOVA) followed by Tokey HDS and LSD. No significant differences were found between the potency of vaccine batches and between macro and micro methods. Reduction in cells sensitivity and potency of vaccines was found with increasing passage number. Significant differences were found between the sensitivity of the cell lines. The highest potency of polio vaccines was obtained using Hela cells (GMT in macro and micro test = 10 6.35); Hep2C cells were afterwards (GMT in macro= 10 6.01 and in micro test= 10 5.94); Vero cells were lowest (GMT in macro= 10 5.78 and in micro test= 10 5.72). So, the sensitivity and accuracy of the potency test for evaluation of the polio vaccine in immunization program in Iran will be assured using the Hela cell line with low passage number in macro and micro methods.