There are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is a newly-discovered one of these peptidases. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and after incubation and the results obtained allowed us to detect if cleavage of the peptides was taking place, and if so, where in the peptide chain. It was found that the enzyme has a preference for another larger, bulky amino acid to follow the proline and that little or no cleavage was observed when polar acidic or other small amino acids were in that location. In terms of the size of the active site, the endopeptidase was found to have optimum activity when there were two more amino acids after the proline, with a fall-off in activity detected for the longer peptides. Data from kinetic studies confirmed the LC-MS results. The methodology described in this paper, which is a combination of LC separation and UV/MS detection, is required for the accurate monitoring of the reactions between the peptidase and its peptide substrates and for analysis of the products of such enzyme-peptide reactions. This work will assist in the design of site-directed inhibitors for new drug therapies.