Free oligosaccharides (fOS) are generated as the result of N-glycoproteins catabolism that occurs in two distinct principal pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N-glycoproteins and the mature N-glycoproteins turnover pathway. The O-(2-acetamidO-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a potent inhibitor of the O-GlcNAcase (OGA) catalysing the cleavage of β-O-linked 2-acetamido-2-deoxy-β-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationaly O-GlcNAc modified proteins. In order to estimate the impact of O-GlcNAc modification on N-glycoproteins catabolism, fOS were analysed by mass spectrometry (MS). MS analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of this population. These abnormal fOS structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal β-hexosaminidases inhibition by PUGNAc. As lysosomal storage disorders (LSD) are characterized by the accumulation of storage material as fOS in lysosomes, our study evokes that the use of PUGNAc could mimic a LSD. This study clearly points out another off target effects of PUGNAc that need to be taken into account in the use of this drug.