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Cytosolic peptide: N -glycanase (PNGase/NGLY1 in mammals), an amidase classified under EC:3.5.1.52, is a highly conserved enzyme across eukaryotes that catalyzes the removal of N -glycans from glycoproteins, converting N -glycosylated asparagine residues into aspartic acid. This enzyme also plays a role in the quality control system for nascent glycoproteins. Despite the development of non-radioisotope-based assay systems such as those using S -alkylated RNase or fluorescent-labeled glycopeptides as substrates, these methods are incompatible with crude enzyme sources, primarily due to the degradation of reaction products by contaminating endogenous proteases. We previously developed an assay system using a 5-carboxyfluorescein-labeled glycosylated cyclo-heptapeptide (5FAM-GCP), a substrate remarkably resistant to endogenous peptidase activity. This system enables the accurate measurement of endogenous NGLY1 activity in various samples, including cell lines, tissues, peripheral blood mononuclear cells, and NGLY1-deficient patient-derived cells, without the interference of proteolytic degradation. We recently advanced this approach by producing a novel fluorescence resonance energy transfer (FRET)-based GCP probe (fGCP) and demonstrated its ability to detect endogenous NGLY1 activity across diverse enzyme sources via fluorescence on multiarray plates. This innovative and straightforward assay now offers reliable disease diagnostics and also allows the measurement of endogenous PNGase/NGLY1 activities across various organisms. Key features • fGCP assay enables measurement of endogenous PNGase/NGLY1 activity in cells and tissues. • An aliquot of 1-5 × 10 6 cells or 50-100 μg of protein extract from tissues is used for this assay. • This assay enables microplate-based real-time measurement of endogenous PNGase/NGLY1 activities. • This protocol requires a fluorescence plate reader equipped with an incubation function., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2025 The Authors; This is an open access article under the CC BY-NC license.)