Background An in‐house Histoplasma urine antigen test for cats might be desirable in certain situations. Objective To validate and compare the diagnostic performance of a monoclonal antibody‐based IMMY urine Histoplasma antigen enzyme immunoassay (IMMY EIA) to the commercially available urine Histoplasma antigen enzyme immunoassay (MiraVista Diagnostics, MV EIA). Animals One hundred ninety‐three urine samples from 105 client‐owned and purpose‐bred research cats. Methods Cats were classified as Histoplasma positive or negative based on diagnostic investigation. The IMMY EIA and MV EIA were performed on all urine samples. Correlation and agreement between the assays were determined. Diagnostic performance was determined and compared between assays. Results The IMMY EIA, with a 0.25 ng/mL diagnostic cutoff, provided a diagnostic sensitivity (DSe), diagnostic specificity (DSp), and diagnostic accuracy (DAc) of 89% (95% confidence interval CI; 73%‐97%), 80% (67%‐89%), and 83% (74%‐90%), respectively. The IMMY EIA, with a 1.1 ng/mL diagnostic cutoff, provided a DSe, DSp, and DAc of 77% (95% CI 60%‐90%), 97% (88%‐100%), and 89% (81%‐95%), respectively. The MV EIA provided a DSe, DSp, and DAc of 94% (95% CI 81%‐99%), 97% (89%‐100%), and 96% (90%‐99%), respectively. Moderate overall agreement was found between MV EIA and IMMY EIA using the 0.25 ng/mL cut‐off (к = 0.44; 95% CI 0.31‐0.57) and the 1.1 ng/mL cut‐off (к = 0.43, 95% CI, 0.31‐0.56). Conclusions and Clinical Importance The IMMY EIA might be useful as a diagnostic test for histoplasmosis in cats. Further modifications of the IMMY EIA are required to achieve the diagnostic performance of the MV EIA.