The use of tissue dissociations to produce single-cell suspensions is foundational to a long history of research applications. More recently, increasingly sensitive and complex down-stream single-cell assays and tissue culture applications are being developed that require refined tissue-specific dissociation protocols that produce consistent, high-quality, single-cell populations. To dissociate tissues, mechanical, enzymatic, and chemical methods are optimized in a tissue-specific manor dependent largely on the histomorphological arrangement of extracellular matrix and intercellular junctions. In the testis where unique junctional complexes form the blood-testis barrier and anchor the germ-line to the seminiferous epithelium, the membranes of interconnected cells are easily damaged, particularly the long processes of Sertoli cells and cytoplasmic lobes of spermatids. The protocol presented is optimized for cell yield, viability, and morphological integrity of singularized testis cells, exhibiting typical viability ≥98% and improved preservation of postmeiotic germ cell morphology. The protocol has been used to map spatiotemporal subcellular dynamics of spermatid-expressed factors in mouse by immunocytochemistry and cell culture applications with pig and mouse dissociated cells. Information intended as a guide is also provided for these downstream assays.