Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)