Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia. Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction. Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining). The wavelengths previously used have shown no phototoxicity on sperm. We used TPLSM to detect spermatogenetic foci in fresh mouse testicular parenchyma without disrupting the tunica albuginea. Fresh surgically retrieved testes were observed using TPLSM within 1 h after extraction. Contralateral testes for each animal were observed using standard histology. Using TPLSM we were able to observe and measure the diameter of seminiferous tubules through the tunica albuginea, similar to the histological control. Structures within epithelial tubules were also observed, although their nature has yet to be identified. TPLSM is a real-time microscopy technique that could detect spermatogenetic foci.