Staining of spermatozoa with the fluorescein-Val-Ala-Asp-fluoromethylketone has already been performed on ejaculated sperm samples, using fluorescence microscopy (FM) or flow cytometry (FCM) in order to score activated caspases. This assay may help in assessing apoptosis and its role in male fertility. The present study compares the above two techniques in order to adopt the most accurate method for detection in human frozen-thawed testicular, epididymal and ejaculated spermatozoa. The analyses were carried out on frozen/thawed testicular (n = 14), epididymal (n = 8) and ejaculated (n = 10) sperm samples. Activated caspases were detected in living spermatozoa using fluorescein-labelled inhibitors of poly-caspases (FLICA). For the measurements by FM, the same-observer and different-observer reliability were assessed in testicular and epididymal sperm samples. The inter-method (FM and FCM) reliability was assessed both in epididymal and ejaculated sperm samples. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements from the same sample were tested by Wilcoxon test for matched pairs. For the same-observer and the different-observer data, the ICC were 0.980 and 0.986. In testicular suspensions, the presence of different types of germinal and somatic cells hampers the differentiation of stained spermatozoa by FCM. For the inter-method reliability, the ICC was 0.903. A lower proportion of the viable spermatozoa stained with FLICA was observed by using FM (−6.60 ± 7.38 %, mean ± SD; p = 0.003) compared with FCM. To measure the proportion of spermatozoa with activated caspases by this test, FM is a highly accurate and reliable method whatever the sperm origin (ejaculate, epididymis, and testis). FCM cannot be used for testicular samples but seems to be more appropriate for analysis of epididymal and ejaculated sperm samples. The systematic lower proportion by FM in measuring the proportion of stained viable spermatozoa with FLICA involves that only the data measured by the same method (FM or FCM) may be compared.