The study of proteins in mammalian cells is critical for the understanding of human health and disease.Currently, there are many methods for detecting proteins in mammalian cells, such as the use of antibodies and adding tags. There is also a desire to study proteins on the endogenous level, such that results reflect the authentic cellular context. It remains a challenge to modify endogenous proteins without adding substantial tags or fusion domains, which might change key characteristics of the target protein. To address this, a “protein editing” technology is described for use in mammalian cells. Protein editing works by using split intein-mediated protein splicing to post-translationally “edit” residuesinto a protein in mammalian cells. By using two pairs of split inteins, the desired editing functionality can be achieved. Briefly, the target protein is tagged with an “Intein Acceptor” sequence at the desired site for editing in mammalian cells, either endogenously or stably overexpressed. In parallel, the corresponding “Intein Donor” containing the payload is generated from E. coli, where non-canonical amino acids (ncAAs) can be robustly incorporated. This recombinant Intein Donor is delivered into mammalian cells containing the Intein Acceptor-tagged protein, at which point splicing will occur, ultimately resulting in the editing of the payload into the target protein. By using a click chemistry handle ncAA, the Intein Donor protein can be labelled with useful small molecules, like fluorophores or biotin, such that these are edited into the target protein in mammalian cells. The protein editing technology was developed and validated using calnexin as a model, with applicationssuch as microscopy or antibody-free isolations of the protein. The technology was then applied to a set of proteins including β-actin, c-Myc, Chk1, and α-actinin, with diverse downstream experiments enabled. The protein editing technology offers several key advantages. First, ncAAs and functional handles can beedited into endogenous proteins. Second, the method is rapid and enables studies of proteins with kinetic control. Third, the method is highly multiplexable across different proteins and labels. In summary, a method for protein editing in mammalian cells has been developed and is poised to enable new discoveries.