An increasing number of nonantibody format proteins are entering clinical development. However, one of the major hurdles for the production of nonantibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics, and genetic knockdowns, we have identified, out of the >700 known proteases in rodents, matriptase-1 as the major protease involved in the degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently, matriptase-1 was deleted in CHO-K1 cells using transcription activator-like effector nucleases (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase (KO) cell line with strongly reduced or no proteolytic degradation activity toward a panel of recombinantly expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next-generation sequencing screening methods, and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.