Although stingless bees have a great potential as commercial pollinators, their exploitation depends on the successful reproduction of colonies on a large scale. To do so, it is essential to develop accurate diagnostic tools that enable a better understanding of the reproductive biology of stingless bees. Sperm counts, sperm morphology and sperm viability (the relative proportion of live to dead sperm), are key parameters assessing semen quality and potential fertilization success. Here we present standardized protocols to assess these three parameters. We used Scaptotrigona aff. depilis (Moure) as a study model. Semen extractions from the seminal vesicles were found to yield better results when performed in mature rather than in younger males. For morphology and viability analyses, the best semen dilution on Hayes solution was adding 120 μl to the contents of the two seminal vesicles. For sperm counts, however, we recommend a higher dilution (1,000 μl). Sperm viability values were higher when Hayes solution was adjusted to pH 8.7, and when samples were analyzed before 24 hours from collection. Based on these results we present standard protocols, hoping they will be useful to future researchers assessing sperm quality in other stingless bee species.