Hydrochloride of 10-{2-hydroxy-3-[N,N-bis-(2-hydroxyethyl)amino]propyl}-2-trifluoromethylphenothiazine (Flu-A) is a analogue of neuroleptic fluphenazine. Flu-A exhibits anti-multidrug resistance, antimutagenic, proapoptopic, and cancer-chemopreventive activities in screening studies. To define identity, quality, and purity of new active substance it is necessary to develop a appropriate analytical method and to establish a degradation profile. Thus, a stability-indicating reversed-phase high-performance liquid chromatography method was developed and validated for quantitative determination of Flu-A in the presence of its degradation products generated under stress conditions. The compound was subjected to oxidation, photolysis, and degradation in aqueous solutions (neutral and acidic), and solid state according to the International Council for Harmonisation Guidelines. The method was also found to be suitable for intermediate and accelerated studies and for the evaluation of kinetic mechanism of Flu-A degradation in aqueous solutions (pH 5.1–7.5, 353 K). The structures of main potential degradation products were established using high-performance liquid chromatography-Electrospray Ionization-mass spectrometry method.