The addition of a single N-acetylglucosamine residue O-linked to serine and threonine residues of nuclear and cytoplasmic proteins is a widespread modification throughout all eukaryotes. The conventional method for detecting and locating sites of modification is a multi-step radioactivity-based protocol. In this paper we show that using quadrupole time-of-flight (Q-TOF) mass spectrometry, modification sites can be identified at a significantly higher sensitivity than previous approaches. This is the first demonstration that sites of O-GlcNAcylation can be identified directly using mass spectrometry.