Sex development is a complex and dynamic process that involves the interaction of many genes, proteins, signaling molecules as well as paracrine and endocrine factors. The initial step in sex development is gonad determination, which in the typical male is the process of development of the bipotential gonad into testis. Subsequently, the testis drive the male sex differentiation through the synthesis of androgens. The disruption of testicular development can result in atypical sex development with increased risk of infertility and tumor development. Therefore, the study of testicular development is crucial in understanding the development of these risks. The recent advances of molecular biology and spatial biology techniques has enabled the characterization of the transcriptome of testicular tissues at a single-cell resolution, giving insights on testicular development and initiation of spermatogenesis. However, these techniques have shown some limitations regarding the isolation of individual Sertoli cells from adult testicular tissues or spatially profiling their transcriptome without contamination from the neighboring germ cells. Here we propose single-nucleus RNA profiling as an alternative technique to characterize the transcriptome of adult testicular tissue at a single-cell resolution. Profiling of RNA in the nuclei rather than in whole cells would enable the more precise characterization of the transcriptome of individual cells, including Sertoli cells, without germ cell contamination. Successful single-nucleus RNA profiling of archived formalin-fixed, paraffin-embedded typical male testis as well as gonadal tissues of individuals with infertility or differences of sex development would help better understanding of the physiological development.