A specific GLC method for the determination of microgram quantities of the central stimulant pemoline in biological fluids is described. Extraction problems due to the low solubility of pemoline are avoided by acid hydrolysis of the drug to 5-phenyl-2,4-oxazolidinedione, which then can be easily isolated by two-phase extraction with dichloromethane, ether, or chloroform. Amide-imide tautomerism enables a cleanup of the extract. Quantitative determination at the microgram level is done on a methylated fraction of the dichloromethane extract by GLC using a suitable internal standard. For supporting evidence of the GLC method's specificity, the compound is also identified by examining an aliquot of the final dichloromethane extract by TLC.