A reversed-phase liquid chromatographic assay was developed for the specific evaluation of metabolic by-products in the urine of galactosemic patients and based on the simultaneous determination of gluconolactone, galactonolactone and galactitol. The procedure involved a lyophilization step and the formation of phenylisocyanate derivatives, followed by injection directly into the chromatograph. Analytical results showed good selectivity, linearity, precision and accuracy. The method enabled the detection of levels as low as 0.05-0.1 ng, and compared favourably with other published techniques for the estimation of aldonic acids in biological fluids.