PrestoBlueTM reagent is a resazurin-based solution currently used as a cell viability indicator by the reducing power of living cells. It has been widely used to assess the viability in different types of cells. However, to our knowledge there is still lack of information describing this technique for the assessment of boar semen quality. Therefore, the purpose of this study was to compare the results between PrestoBlueTM assay, flow cytometry (FC) and computer-assisted sperm analysis (CASA), in order to establish the protocol of PrestoBlueTM assay for boar semen. Four Duroc-Meishan crossbred boars of 18-26 mo of age were used in this study. Boar semen was collected weekly and divided into live and killed portions. The live portion was kept at room temperature, and the killed portion was plunged into liquid nitrogen and thawed at 37 °C for two cycles. The two portions of semen samples were diluted to 300 and 150 × 106 sperm/ml with PBS and then mixed to make semen aliquots of live and killed sperm ratios of 0/10, 2/8, 4/6, 6/4, 8/2, and 10/0 (v/v), respectively. Each sample was analyzed by (1) PrestoBlueTM assay: volume (µL) of semen/PrestoBlueTM were 100/10, 200/10 and 200/20; OD values were detected at 0, 10, 20, 30, 40, 50, 60 min, (2) FC for sperm viability, acrosome integrity and mitochondria activity, and (3) CASA for total and progressive motility. The results indicated that the most optimal boar semen concentration and volume of semen/PrestoBlueTM were 300 × 106 sperm/ml and 100/10, respectively. The PrestoBlueTM reduction rates at 30 min are highly correlated to sperm viability (R=0.969), acrosome integrity (R=0.972), mitochondria activity (R=0.921), total motility (R=0.952) and progressive motility (R=0.963); and the correlation coefficients increased with the prolonging of incubation time. Based on our findings, PrestoBlueTM assay can be an efficient and economical method to evaluate boar semen quality.