The combination of liquid chromatography coupled to mass spectrometry (LC-MS) and acute toxicity studies with Daphnia magna was used to elucidate the water stability of the cytostatic compounds, mitoxantrone and chlorambucil, and their transformation products. Both compounds were rapidly degraded in water with the subsequent formation of bioactive transformation products. Mitoxantrone suffered a rapid change in its conformation with the formation of four toxic transformation products, which were unaltered and stable in water along the two day studied period. LC-MS analyses allowed to identify the conformational changes of mitoxantrone that included the loss of the two amino alcohols (N-ethylethanolamina) [C4H10NO]+, the loss of N-ethylethanolamina [C4H10NO]+ and N-methylethanolamina [C3H8NO]+, the loss of CH2OH from the original molecule and the formation of mitoxantrone dicarboxilic acid. Chlorambucil was also rapidly degraded in water loosing an hydroxyl group and forming a bioactive transformation product that further degraded within the following 12 hours. The degradation of chlorambucil was also related with an exponential loss of toxic activity towards Daphnia magna survival. The present results indicate that LC-MS methods should target transformation products since field concentrations of these compounds measured in water may not reflect their toxicity.