Two simple, selective and sensitive spectrophotometric methods are described for the determination of 6-aminopenicillanic acid (6-APA). The methods are based on the reaction of 6-APA with either bromophenol blue (BPB) or bromothymol blue (BTB), to give orange-red and green species, respectively. The coloured products are quantified spectrophotometrically at 625 and 616 nm for BPB and BTB, respectively. The optimization of the different experimental conditions is described. No interferences from different -lactams and common degradation products were observed in the determination of 6-APA using BTB, while flucloxacillin, dicloxacillin, adrenaline, vitamin C, urea and common degradation products in any percentage interfere on using BPB only. The BTB method was better than the BPB method, because of its wider range of determination (0.4–20 g ml–1 vs. 0.4–7.2 g ml–1 on using BPB), higher molar absorptivity and Sandell sensitivity (3.27 103l mol–1 cm–1 and 0.099 g cm–2 vs. 2.82 103lmol–1 cm–1 and 0.115 g cm–2), greater stability (3 and 10 days on using BTB and BPB, respectively) and better selectivity. The results were compared with those given by the Official United States Pharmacopeial XXI method.