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Separation of the amino acids in urine by use of thin-layer chromatography (TLC) has hitherto required that the specimen be first desalted and then chromatographed in two dimensions with at least two pairs of developing solvent systems. We wished to simplify both steps. The customary method of desalting on a column is replaced by desalting on a plate that supports a strongly acid cation-exchanger and a cellulose layer. This method, originally developed for one-dimensional TLC, is used here for two-dimensional TLC. Urine is applied to the ion-exchange layer and strong acids and neutral substances are removed with water. The amino acids are then chromatographed into the cellulose layer, and are separated there two dimensionally with a newly devised pair of developing solutions. This pair of solvents separates nearly all of the amino acids in urine.