Parameters are described for the qualitative and quantitative analysis of a mixture of isoniazid and its acetyl derivative. The compounds are chromatographed on an octadecylsilane column, using absolute methanol-distilled water (60:40) at pH 2.5 containing 0.01 M dioctyl sodium sulfosuccinate. The flow rate was 2.0 ml/min (2500 psig). The separation and quantification are applicable to plasma and urine samples. The determination in each biological fluid can be achieved in approximately 90 min with percentage accuracies for isoniazid of 5.25 and 7.45 and for the acetyl derivative of 4.47 and 1.56 in plasma and urine, respectively.