A novel and sensitive high-pressure liquid chromatography (HPLC) method was developed for the simultaneous estimation of Imiquimod (IMI) and Thymoquinone (THY). Chromatographic elution was accomplished using Hypersil™ C-18 reverse phase column measuring 250 mm × 4.6 mm (5 µm) and mobile phase as Acetonitrile: Water (0.1% formic acid) in 65:35 ratio. The flow rate used in the method is 1.0 mL/minute and was observed at 249 nm employing PDA Detector. The run time of the analytical procedure was 16 minutes. The retention time was obtained at 4.1 min (IMI), and 14.3 min (THY). International Conference on Harmonization (ICH) guidelines were followed to validate the method and the parameter included a limit of detection (LOD), robustness, limit of quantification (LOQ), and system suitability. The regression graph plotted showed linearity from 10 to 100 μg/ml concentration. All results are under acceptable limits and the method could be suitably employed for the simultaneous estimation of both drugs in quality control and assay.