Potentiometric and spectrophotometric pH-titration of the multiprotic cytostatics bosutinib for dissociation constants determination were compared. Bosutinib treats patients with positive chronic myeloid leukemia. Bosutinib exhibits four protonatable sites in a pH range from 2 to 11, where two pK are well separated (ΔpK > 3), while the other two are near dissociation constants. In the neutral medium, bosutinib occurs in the slightly water soluble form LH that can be protonated to the soluble cation LH43+. The molecule LH can be dissociated to still difficultly soluble anion L−. The set of spectra upon pH from 2 to 11 in the 239.3–375.0 nm was divided into two absorption bands: the first one from 239.3 to 290.5 nm and the second from 312.3 to 375.0 nm, which differ in sensitivity of chromophores to a pH change. Estimates of pK of the entire set of spectra were compared with those of both absorption bands. Due to limited solubility of bosutinib the protonation in a mixed aqueous-methanolic medium was studied. In low methanol content of 3–6% three dissociation constants can be reliably determined with SPECFIT/32 and SQUAD(84) and after extrapolation to zero content of methanol they lead to pKc1 = 3.43(12), pKc2 = 4.54(10), pKc3 = 7.56(07) and pKc4 = 11.04(05) at 25 °C and pKc1 = 3.44(06), pKc2 = 5.03(08) pKc3 = 7.33(05) and pKc4 = 10.92(06) at 37 °C. With an increasing content of methanol in solvent the dissociation of bosutinib is suppressed and the percentage of LH32+ decreases and LH prevails. From the potentiometric pH-titration at 25 °C the concentration dissociation constants were estimated with ESAB pKc1 = 3.51(02), pKc2 = 4.37(02), pKc3 = 7.97(02) and pKc4 = 11.05(03) and with HYPERQUAD: pKc1 = 3.29(12), pKc2 = 4.24(10), pKc3 = 7.95(07) and pKc4 = 11.29(05).