The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi glycan-modifying enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by endogenous SPPL3 and released into conditioned cell culture supernatants.