An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and working range, limits of detection and quantification, accuracy and recovery. Samples taken at various stages of Reovirus purification were used for the validation of the method. The method was specific for Reovirus which eluted around 9.8min without interference from any other component in the sample. Reovirus can be detected between 0.32E+12 and 2.10E12VP/mL by the proposed method that has the correlation coefficient of linearity equal to 0.9974 and the slope of linearity equal to 5.74E-07 area units/(VPmL).