Microalbuminuria is a marker of diabetic nephropathy and cardiovascular risk. Immunoassays underestimate the amount of intact albumin present in urine. The purpose of this study was to evaluate a new urinary albumin assay that uses size exclusion high-performance liquid chromatography (HPLC). We determined the limit of detection, linearity, imprecision, a comparison with an immunoturbidimetric assay, and pediatric and adult reference intervals. The limit of detection was 3.4 mg/L. The assay was linear from 4 to 240 mg/L. Total imprecision was less than 10% from 16 to 206 mg/L. Comparison of the albumin/creatinine ratio by HPLC with an immunoturbidimetric method showed positive proportional bias, which decreased with increasing concentrations of albumin. Nonparametric reference intervals were 22 to 250 mg/g for girls, 20 to 130 mg/g for boys, 14 to 62 mg/g for women, and 10 to 37 mg/g for men. This HPLC assay for urinary albumin shows acceptable performance and quantifies albumin species that are not detected by immunoassay. Separate reference intervals for children and adults seem necessary.