Several studies have shown that sperm viability (SV) is a key trait during sexual competition. However, this has not yet been tested in spiders as no protocol has been developed to quantify SV. Here, we describe a methodology for estimating SV using the pholcid spider Holocnemus pluchei (Scopoli, 1763). In this method, male spermatozoa were released from the sperm ducts of copulatory bulbs in 50 µl saline solution and mixed with a vortexer. Finally, 10 µl of the sperm solution were combined with 1 µl of GelRed stain. We counted a total of 200 cells and calculated the percentage that was not stained. Males showed almost 100% of live sperm inside their genital bulbs. GelRed stain demonstrated a high effectiveness to distinguish between dead and living sperm cells in spiders and it is thus a more reliable option compared to the dyes typically used, such as propidium iodide and sybr14. The high SV in H. pluchei may be explained by the multiple mating nature of females, which suggests a selection for enhanced effectiveness during sperm competition within the female genital tract.