Publisher Summary This chapter discusses oxygen-dependent asparagine hydroxylation. Asparagine hydroxylation occurs adjacent to the C-terminal transcriptional activation domain (CAD) of the Hypoxia-induced factor (HIF), repressing transactivation by blocking the interaction of HIF with the transcriptional coactivator p300 and prevents activation of transcription. Under hypoxic stress, the hydroxylases are inactivated and HIF is stabilized and activated transcriptionally, resulting in specific gene induction. Hydroxlation of asparagine and aspartic acid residues has been observed previously within consensus sequences of EGF-like domains of several proteins, but no direct biological function has been attributed to these modifications. This chapter discusses methods for detection, characterization, and assay of oxygen-dependent hydroxylation of asparagine residues. In particular, the role of modern mass spectrometry methods in characterizing modification in HIF is highlighted. Detection and characterization of protein-bound hydroxyasparagine , demonstration of a hydroxylation site between residues 846 and 864 of HIF-2α 774–874 dependent on the oxygen environment of mammalian cells, and characterization of asparagine 851-specific hydroxylation of HIF-2α CAD in mammalian cells are discussed