Measuring mRNA abundance at the level of the whole transcriptome has become an indispensable tool for the investigation of fundamental biological processes in higher eukaryotes. However, traditional transcriptomic methods are destructive and ignore the three-dimensional organization of cells within tissues and organs. Spatial transcriptomics is a cutting-edge technology that begins to address this limitation and facilitates transcriptomic analyses within the normal spatial context of two-dimensional tissue sections. This method permits a comprehensive appreciation of cellular heterogeneity according to spatial organization and overcomes the critical limitation of traditional bulk methods by preserving tissue architecture. Spermatogenesis is an exemplary cell lineage that varies in a spatial manner. Specifically, the stages of the cycle of the seminiferous epithelium elaborate the cell type variation along the length of seminiferous tubules, which are revealed in two-dimensional space as distinct cellular associations in seminiferous tubule cross-sections. Interpreting the wealth of available single-cell RNA-seq data requires appreciation of cycle stage, which can only be accomplished by understanding each germ cell’s spatial context. In this chapter, we provide an overview of spatial transcriptome technologies and step-by-step guidelines for application of one such method from Curio Biosciences, Seeker (SlideSeq V2) to the testis. The text elaborates on testicular tissue preparation and quality assessment, section and spatial transcriptome library preparation, and bioinformatic methods for data analysis using a simple exemplary analysis of round spermatids in the mouse testis.