Male infertility is a complex multifactorial disease whose underlying etiology remains unknown. Despite substantial efforts, 60–75% of the cases are classified as idiopathic, which makes them a subject of empirical and generalized treatments. This puts the impetus on a comprehensive understanding of the mechanisms of spermatogenesis and testis function thereby providing approaches to molecular diagnosis and targeted treatments. Azoospermia, characterized by the complete absence of sperm cells in seminal fluid, is one of the known conditions resulting in male infertility. To elucidate the complex molecular mechanisms causing male azoospermia, an approach combining high-resolution shotgun proteomics with imaging mass spectrometry (IMS) was carried out on testicular specimens from patients with mixed atrophy (MA) and spermatogenic arrest in spermatocyte 1 (SA). The obtained data showed dysregulation of spermatogenesis and emergence of hemostasis and pro-inflammatory processes in both testicular phenotypes. Furthermore, the IMS analysis revealed inter-patient and inter-tubular variability in the MA samples along with the region-specific dysregulation of basic cellular functions in both SA and MA samples. In conclusion this approach enabled to identify the molecular processes resulting in male infertility along with cell types and/or the tissue compartments responsible for it. It would potentially be instrumental in assessment of diseases and formulation of treatment strategies within tissues with complex and heterogeneous structure.