Evaluation of renal hemodynamics requires estimation of effective renal plasma flow, which is commonly measured by the renal clearance of p-aminohippuric acid (PAH). There are many existing methods for PAH assay that are complicated, expensive, or time consuming. We describe a rapid, precise, and accurate microplate-based assay of PAH using p-dimethylaminocinnamaldehyde, which produces a red color on reaction with PAH, and compare it with a reference HPLC method. Renal PAH clearances were measured in 10 volunteers, and clearances were calculated by using the new and HPLC methods. There was excellent agreement between the HPLC and the microplate method of PAH assay. The average ratio of microplate to HPLC method was nearly 1.0, and the limits of agreement (2 SD) for plasma, urine, and clearances were 17.2, 19.3, and 25.5%, respectively. Intraday coefficient of variation for urine and plasma was <7%; interday coefficient of variation was <6% for urine and plasma samples. The microplate method is a reliable alternative to a reference HPLC method and can be performed for a fraction of the cost, time, and reagents.