<p>Arginase is widely used to analyze nitric oxide-related physiology and inflammation. However, traditional activity assays are often complicated and lack isoform resolution because of the shared substrates and products of arginase 1 (ARG1) and arginase 2 (ARG2). We developed a bead-based assay that selectively measures ARG1 activity by capturing ARG1 on anti-ARG1-coated magnetic beads, followed by on-bead catalysis and a simple colorimetric measurement of urea. Methodologically, we assessed the analytical performance of the assay using recombinant enzymes and human serum. Within the evaluated range, the assay demonstrated linear responses and reproducible readouts across three independent replicates, with variability quantified by SD and CV%. Immunoblotting following bead pretreatment confirmed ARG1-specific capture, with no detectable adsorption of ARG2, thereby confirming selectivity. Further analysis of antibody-enzyme interactions revealed that the anti-ARG1 monoclonal antibody (clone 6G3) did not inhibit ARG1 catalysis, ensuring that its capture did not modify enzymatic activity. In human serum with inflammation, nonparametric Spearman analyses indicated that total arginase activity was closely correlated with ARG1 activity, whereas the abundance of ARG1 or ARG2 proteins alone showed a limited association with activity. These data collectively suggest that the enzymatic activity of circulating arginase in the serum is primarily attributed to the ARG1 component and that protein abundance does not accurately reflect functional capacity. The proposed bead-capture assay offers a simplified workflow, eliminating the need for endogenous urea depletion or radioisotopes, and facilitates isoform-specific activity measurements to support translational research. Further validation in well-characterized patient cohorts will be required to establish clinical utility.</p> Graphical abstract <p></p>