Non-obstructive azoospermia (NOA) is the most difficult form of male infertility to manage. It usually requires sperm retrieval from the testis, which is most challenging due to sperm rarity. Here, we describe the recovery of testicular sperms that had been missing and whose original retrieval results were negative. Salvage microsurgical testicular sperm extraction and sperm testing were performed on a 36-year-old male with NOA. Neither in the operation room nor after an inspection in the embryology laboratory were any sperm detected. The obtained tissue was advised to be frozen because the patient data and surgical microscopy predicted a favorable outcome, and the tissue processing was done in an inappropriate environment. About 1 month later, the specimen was thawed, crushed, and re-examined. Successful oocyte fertilization resulted from an effective detection of sperms and their direction to intra-cytoplasmic sperm injection. This is the first case report that, to the best of our knowledge, describes the stepwise laboratory processing of testicular tissue and its capacity to recover lost sperms in challenging NOA cases and under less-than-ideal working conditions.