A high performance liquid chromatographic method for simultaneous determination of isoniazid and acetyl isoniazid in urine was developed. Urine samples were filtered through Whatman number 1 filter paper and diluted filtrate was directly injected into C8 column (250mm). The mobile phase comprised of water and methanol in the ratio of 85:15. The analytes were detected at a wavelength of 274nm. The assay was specific for isoniazid and acetyl isoniazid and linear from 1.25 to 40.0μg/ml for isoniazid and 3.125 to 100μg/ml for acetyl isoniazid. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of isoniazid and acetyl isoniazid from Urine was 100% and 95% respectively. Due to its simplicity, the assay can be used for determination of isoniazid acetylator status using urine and to check patient adherence to tuberculosis treatment.