In this study, a simple spectrophotometric method was developed for the quantitative determination of rifampicin in aqueous solution. The method is based on the property of rifampicin to modify, in aqueous media at pH of 7.0, the degree of ionization of the functional groups. After ionization, a coloured compound is formed, which can be analysed spectrophotometrically. The visible spectra of the yellow compound were recorded at pH of 7.0, in different buffer solutions (phosphate, hexamethylentetraamine (HMT) and borax) and the qualitative characteristics were determined. The highest absorbance was obtained in phosphate buffer, at λ = 470 nm, against distilled water, and these conditions were selected for quantitative determinations. This procedure allows the quantitative analysis of rifampicin in a wide concentration range (0.01 - 0.16 mmol·L-1), with a detection limit of 0.0002 mmol·L-1. The very good correspondence between the calculated and experimental values of rifampicin concentration shows that this method is valid and can be used for quantitative analysis, at least for laboratory studies.