It is possible to produce anti-phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross-react with either the cognate unphosphorylated protein or other phosphoproteins. Unlike conventional antibodies, anti-phosphopeptide antibodies provide information regarding not only the abundance of a protein but also its activity. Also, unlike general anti-phosphoamino acid (e.g., anti-phosphotyrosine) antibodies, which have broad reactivity, anti-phosphopeptide antibodies may have unique specificity toward the cognate proteins. Such reagents not only facilitate conventional in vitro analysis of phosphoproteins, but also allow heretofore impossible applications, e.g., differential isolation of species of a particular protein that have been phosphorylated at individual phosphorylation sites, as well as analysis of the functional state of a protein in situ by immunohistochemical techniques. This unit provides protocols for the production of both polyclonal and monocloncal anti-phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to the affinity matrix (Affi-Gel 10); BSA-agarose affinity matrix is commercially available.