Histochrome is the medicinal form of echinochrome (2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoquinone). The drug pharmacodynamics and its relationship with metabolism, which was revealed in previous investigations, predetermined the aim of this work: to study excretion of echinochrome and its possible metabolites in urine of rats. Histochrome was introduced to Wistar rats (n = 10) subcutaneously in a dose of 10 mg/kg. Naphthoquinone derivatives were extracted from the acidified urine by ethyl acetate and analyzed by high performance liquid chromatography with UV detection (HPLC/UV) and mass-spectrometric detection (HPLC/MS). It was established that histochrome is completely metabolized in an organism and excreted by kidneys in the form of 3-methoxy-2,5,6,8-tetrahydroxy-7-ethyl-1,4-naphthoquinone and 2-metoxy-3,5,6,8-tetrahydroxy-7-ethyl-1,4-naphthoquinone.