A colorimetric method for the determination of ampicillin (Amp.) and 6-aminopenicillanic acid (6-APA) are described, based on the reaction of these drugs with acenaphthenequinone in basic media to give a highly intense red coloured product. The latter exhibits an absorption maximum at 610 nm with apparent molar absorptivities of 2.83 and 1.45 × 104l. mol−1cm−1and Sandell sensitivities of 0.013 and 0.015 μg cm−2for Amp. and 6-APA, respectively. The optimum concentration ranges are 0.4-10 and 0.4-14 μg ml−1for Amp. and 6-APA, respectively. For more accurate results, Ringbom optimum concentration ranges are 1–8.5 and 1–12 μg ml−1for Amp. and 6-APA, respectively. Statistical analysis indicated that there was no significant difference between the results obtained by the described method and those of the official methods. The mean recoveries percentage were found to be 99.5 × 1.1% for pharmaceutical formulations and 99.1 × 1.6% for serum and urine samples. The method is selective for the determination of Amp. or 6-APA in the presence of their degradation products, additives and excipiences that are normally encountered in dosage forms. The proposed method was applied successfully to the determination of Amp. in pharmaceutical formulations. Also, applicability of the proposed method to human serum and urine is presented and the validity assessed by applying the standard addition technique.