Characterization of how a stimulus regulates the dynamics of protein-protein interaction is critical for understanding how a particular protein is regulated in an intracellular signaling network. Protein hydroxylation, which is a posttranslational modification catalyzed by oxygen-dependent enzymes, is a crucial regulator of protein-protein interactions. Under low oxygen conditions, the activity of many hydroxylases is inhibited, which results in a reduction of substrate hydroxylation. These changes alter the interactome of the substrate, and this dynamic rewiring of signaling networks explains crucial aspects of the adaptive response to hypoxia. In order to fully understand the systemic role of hydroxylation, it is necessary to identify a comprehensive set of substrates, as well as to determine which residues are hydroxylated. In addition, hydroxylation-dependent changes in the interactome of the substrates are indicative of the molecular function of the modification. To identify new substrates of hydroxylases, we have developed an approach involving the use of a pharmacological substrate-trap strategy followed by label-free quantitative mass spectrometry. An overview is provided for the sample preparation, mass spectrometry techniques, and statistical analysis used for detection of new substrates, hydroxylated residue, and hydroxylation-dependent protein-protein interaction changes.