<p>A green method for simultaneous determination of water soluble vitamin (vitamin C) and fat soluble vitamin (vitamin A) was developed using reversed phase high performance liquid chromatography technique. The method succeed to separate the water-soluble and fat-soluble vitamins by isocratic elution using Agilent Zorbax octylsilane column (250 × 4.6&#xa0;mm, 5&#xa0;μm) in a short single run. The proposed mobile phase consisted of buffer (10 mM potassium dihydrogen phosphate and 3 mM hexane sulfonic acid sodium salt), pH adjusted to 2.5 using orthophosphoric acid and methanol in a ratio (8:92 v/v) with flow rate 1.0 mL.min^− 1 and UV detection 328&#xa0;nm for vitamin A and 243&#xa0;nm for vitamin C in concentration range (0.5–30 IU.mL^− 1) and (1–60&#xa0;µg.mL^− 1), respectively. Accuracy results were 99.49% ± 1.58 for vitamin C and 100.26% ± 1.86 for vitamin A, limit of detection (L.O.D) of vitamin C is 0.3&#xa0;µg.mL^− 1 while for vitamin A is 0.15 IU.mL^− 1 and limit of quantification (L.O.Q) of vitamin C is 1.0&#xa0;µg.mL^− 1 while for vitamin A is 0.5 IU.mL^− 1. Analytical eco scale and green analytical procedure index showed that our proposed method is greener than the reported method. The proposed method validation was performed according to ICH guidelines and the method was applied successfully for determination of vitamin A and vitamin C simultaneously in cosmetic nano-formulation, pharmaceutical dosage form and in pure forms.</p>