A sensitive, reliable, and stable high performance liquid chromatographic (HPLC) methodology established further determination by combination with Carvedilol (CVD) and 5-Fluorouracil (5-FU) in innovative nano vesicular transethosomal drug delivery system. Method development was performed by a Box-Behnken QbD to systematically achieve optimal chromatographic performance with adequate resolution and reduced analysis time. A Target Analytical Profile (TAP) was utilised to determine critical parameters that affecting its quality attributes. The percentage of organic solvent, mobile phase pH and buffer strength were recognized as key influential variables. QbD design mainly evaluate critical analytical space along with an appropriate control strategy. A Kromasil C18 column was used to separate the samples efficiently. We estimated final mobile phase 60:40 ration with its flow rate 1mL/min at Isosbestic point at 254 nm. Developed method had very strong linear responses for CVD in the series dilution of 50-150 μg/mL and for 5-FU in the range of 100 -300 μg/mL. The parameters for measuring CVD were 3.73 and 11.31 µg/ml, and the limits for finding and measuring 5-FU were 6.08 and 18.43 µg/ml. Validation results met FDA standards, and forced degradation studies showed that the method could demonstrate the difference between analytes and their degradation products. This means that it is suitable for advanced cancer therapy applications that require high precision.