Mammalian sex determination is a highly complex developmental process that is particularly difficult to study due to the limited number of gonadal cells present at the bipotential stage, the large cellular heterogeneity in both testis and ovaries and the rapid sex-dependent differentiation processes. Single-cell RNA-sequencing (scRNA-seq) circumvents the averaging artifacts associated with methods traditionally used to profile bulk populations of cells. It is a powerful tool that allows the identification and classification of cell populations in a comprehensive and unbiased manner. In particular, scRNA-seq enables the tracing of cells along developmental trajectories and characterization of the transcriptional dynamics controlling their differentiation. In this review, we describe the current state-of-the-art experimental methods used for scRNA-seq and discuss their strengths and limitations. Additionally, we summarize the multiple key insights that scRNA-seq has provided to the understanding of mammalian sex determination. Finally, we briefly discuss the future of this technology, as well as complementary applications in single cell -omics in the context of mammalian sex determination.