Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia inducible factor (HIF), interest has centred on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the I(kappa)B and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Whilst these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine, due to the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological “substrate trapping” together with stable isotope labeling of amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined thirteen new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat, and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated and detailed studies of one, Tankyrase-2 revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the circa 300 ARD-containing species in the human proteome.