The purpose of the present study was to formulate as well as validate a new “green” “HPLC-UV” method for rapid quantification of doxorubicin (DOX) in standard drug, marketed products, and bacterial ghost matrix. High-performance liquid chromatography (HPLC) study was carried out at room temperature ( $$25\,{^{\circ }}$$ C) using a Nucleodur 150 $$\times $$ 4.6 mm RP C8 column filled with 5 $$\upmu $$ m filler as a static phase. The mobile phase consisted of methanol: ethyl acetate (7:3% v/v) which was delivered at a flow rate of 1.0 mL/min and the drug was detected in UV mode at 480 nm. The proposed technique was validated for linearity, selectivity, accuracy, precision, robustness, sensitivity as well as specificity. The excellent linearity was obtained between the linear regression data for the calibration plots ( $$r^{2} \,0.9986$$ ). LOD and LOQ were calculated and found to be 0.0325 and 0.0985 $$\upmu $$ g/mL, respectively. Satisfactory values were obtained by the statistical treatment of different validation parameters. The utility of the proposed process was confirmed by analysis of DOX in bacterial ghost matrix and marketed products. The newly developed “green” HPLC-UV system effectively determined DOX peak along with its degradation products which established the stability-indicating property of the method. These outcomes indicated that the developed HPLC method could be effectively used for routine investigation of DOX in standard drug, various pharmaceutical formulations, and drug release samples.