1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) assay, flow cytometry (FC) or computer-assisted spermatozoa analysis (CASA) were compared. 2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60, and 30 × 106 cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10, 20, 30, 40, 50, 60 min, (2) viability with flow cytometry, and (3) motility with CASA. 3. The WST-8 reduction rate was closely correlated with spermatozoa viability and motility. The optimal semen concentration for the WST-8 assay was 120 × 106 cells/ml, and the standard curves for spermatozoa viability and motility predictions respectively were yviability60 = 162.8x + 104.96 (R2 = 0.9594) after 60 min of incubation and ymotility40 = 225.09x + 96.299 (R2 = 0.8475) after 40 min of incubation. 4. It was conclused that the WST-8 assay is useful for the practical evaluation of cockerel spermatozoa viability and motility. Compared to FC and CASA, the WST-8 assay does not require expensive and complex instrumentation in the lab. Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa viability and motility at the same time which all lead it to be efficient and economical for semen quality assessment.