The anthracycline antibiotic doxorubicin is utilized in the clinical treatment of many type of cancers. Doxorubicin for injection is a lyophilized red powder and was highly soluble in the solvent system utilized in this study for spectrophotometric and HPLC determination (77.7% ethanol, 0.0068 molar acetic acid, and 22.3% water). An absorbance spectrum was obtained for doxorubicin in the solvent system utilized between 325 nm to 850 nm. A strong absorbance peak was found between about 430 nm and 550 nm, with the peak apex occurring at 480 nm. The spectrophotometric determination of doxorubicin was accomplished at 480 nm using 1.0 cm glass cuvettes and a Spectronic 21D spectrophotometer. Doxorubicin dissolved quickly into the solvent having ethanol, acetic acid, and water. The calculated molar absorption coefficient of doxorubicin at 480 nm is 2244.0 Liter/mole•cm. This study achieved a linear set of standards for doxorubicin assay at concentrations from 0.100E-04 molar to 1.91E-04 molar doxorubicin. The linear relationship is defined by y = 0.2148x - 0.0016, with Pearson r correlation of 0.9999 (coefficient of determination R² = 0.9999).  Likewise, for HPLC determination of doxorubicin at 320 nm, linearity of standards was obtained from 0.0500E-04 molar to greater than 2.00E-04 molar (y = 277.47x - 4.4172) with Pearson r of 0.9997 and 99.94% of the variance is explained by model (coefficient of determination R² = 0.9994). The analyte doxorubicin was highly soluble in a solvent mixture that was suitable for assay by spectrophotometer and reversed phase HPLC.