Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by α 2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α 2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α 2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of∼ 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α 2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to α 2-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α 2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.