In this study, a multiwavelength spectrophotometric method was used for the determination of the dissociation constants of drugs used for treatment of colorectal cancer, folinic acid (leucovorin), 5-fluorouracil, and irinotecan, in water and acetonitrile water binary mixtures up to a 0.50 volume fraction at 25 degrees C and an ionic strength of 0.1 mol . L-1 KCl. The absorbance/pH profiles were assessed and found to conform to those of polyprotic acids. Data evaluation was performed using the STAR software program which calculates stability constants and molar absorbances of the pure species by multilinear regression. A linear relationship between acidity constants and the mole fraction of acetonitrile in the solvent mixture was observed. The effect of solvent properties on acid-base behavior is discussed.