Yellow fever is a disease caused by the prototype virus of the genus Flavivirus and remains endemic in tropical forest regions from Africa and South America, despite the availability of effective vaccines. These are capable of inducing a rapid specific immune response, with the formation of neutralizing antibodies that appear early, are protective and long lasting. The Plaque Reduction Neutralization Test is considered the most sensitive and specific test for quantification of neutralizing antibodies, and the reference method for assessing the protective immune response after vaccination. This study evaluated the reliability (repeatability and reproducibility) and accuracy (sensitivity, specificity and overall accuracy) of micro-PRNT50 and compared its performance with the micro-PRNT90. Although the micro-PRNT50 has showed satisfactory levels of reliability (ICCs ranged from 0.62 to 0.NorNormas e Manuais Técnicosas e Manuais Técnicos6 for repeatability and 0.72 for reproducibility) and accuracy (sensitivity of 91.1%, specificity of 72.9% and overall accuracy of 78%), the micro-PRNT90 showed higher performance, with ICCs for repeatability ranged from 0.78 to 0.79 and 0.81 for reproducibility, sensitivity of 100%, specificity of 94.7% and overall accuracy of 95%. Modifications in the test methodology and changes in the classification criteria in the readings of the results obtained will be important to improve the accuracy of micro-PRNT.