5-Fluorouracil (5-FU) is a conventional anti-cancer agent. Several studies have shown the synergism effect of Cannabidiol (CBD) along with 5-FU for skin cancers, thus making this combination cogitate for researchers for the management of skin cancer. A novel, economical, sensitive and robust high-pressure liquid chromatography (HPLC) technique was established for the evaluation of 5-Fluorouracil (5-FU) and Cannabidiol (CBD) simultaneously. Chromatographic elution was done using the stationary phase Hypersil™ C18 reverse phase column 250 mm × 4.6 mm (5 µm) and employing mobile phase as Methanol: Water in ratio 90:10 running at a flow rate of 1.0 mL/min and evaluated at 235 nm. The total run time of the proposed technique was 4 min. The retention time was obtained at 1.43 min (5-FU), and 2.27 min (CBD). The proposed analytical methodology validation was executed in consistent with International Conference on Harmonization (ICH) regulations and the validation parameter taken into consideration were the limit of detection (LOD), robustness, the limit of quantification (LOQ), and system suitability. A linear standard curve was plotted from the 10 to 100 μg/ml concentration range. The LOD of the proposed method was 4.75 ng/ml for 5-FU and 12.12ng/ml for CBD. All results are under acceptable limits and the method could be suitable for employed in the synchronous determination of sample drugs in quality control and assay