We developed a fully automated quantitative immunoassay for the detection of prostaglandin E-major urinary metabolite (PGE-MUM). In this study, we evaluated the analytical performance of this assay. Sensitivity, within-run reproducibility, correlation with radioimmunoassay (RIA), cross-reactivity, dilution linearity, spike recovery performance, analyte stability, and effects of coexisting substances were evaluated. The assay was also used to measure PGE-MUM in 211 healthy people. The limit of detection and quantification were 1.0 and 1.3 ng/mL, respectively. When the assay was performed six times in a single run, the coefficient of variation ranged from 1.4% to 2.2%. The coefficient of correlation with a preceding RIA method was 0.970 with a correlation slope of 0.88. There was no cross-reactivity with PGE-MUM analogs. Linearity of dilution was confirmed at up to 16-fold dilution with assay results within 100 ± 20% of the theoretical values calculated based on the undiluted sample. Spike recovery was good and ranged from 94% to 101%. Analyte stability was tested by storing samples at 25°C for 6 days, 10°C for 1 month, and by performing up to five freeze-thaw cycles. Assay results were all within 100 ± 10%, the values measured before storage and before the freeze-thaw process. Assay results in healthy people ranged from 3.1 to 162.7 ng/mL (mean: 35.8 ng/mL). After correction for creatinine, the 95% confidence interval was 8.68-42.25 μg/g creatinine. The assay precisely detects PGE-MUM.