Abstract EO9 (apaziquone) is a novel, promising anticancer agent, which is currently being investigated for the intravesical treatment of bladder cancer. EO9 contains a highly reactive aziridine ring in its structure that limits its chemical stability in acidic aqueous solutions. The stability of the pharmaceutically formulated EO9 in human urine, including the effects of several parameters such as temperature, buffer strength and pH have been investigated. Urine extracts were analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC–MS/MS) using a TurboIonspray interface and positive-ion multiple reaction monitoring. EO9 was unstable in urine at 43 °C during the instillation for longer than 1 h. However, the drug was stable in human urine for 3 h at 37 °C. EO9 is stable in urine stabilized with TRIS buffer (pH 9.0; 5 mM) for up to three freeze/thaw cycles at −20 and −70 °C and 3 months of storage at −70 °C. The results also illustrated that with the lower pH in urine, EO9 became more unstable. Furthermore, a new degradation product of EO9 was discovered and successfully identified as EO9-Cl. The outcomes of these stability experiments will be implemented to insure proper sample handling at the clinical sites, transport, storage, and sample handling during analysis in the forthcoming preclinical studies of EO9 in superficial bladder cancer, supported by bioanalysis and pharmacokinetic monitoring.