Background: VACUETTE® Blood Collection Tubes are used for the collection of venous blood. VACUETTE® tubes are used to collect, transport, store and process blood for testing serum, plasma or whole blood in the clinical laboratory for professional use. Objective: Evaluate the effect of blood specimen collection methods of the VACUETTE Serum Sep Clot Activator tube (gel tube), in determination vitamin A by HPLC. Methods: Blood specimen was divided into three parts, Three tubes, for Part I, No Additive tubes were drawn, for Part 2 Serum SepClot Activator tube (gel tube), and for Part 3 EDTA-coated plastic tubes. The tubes were gently mixed using ten complete inversions immediately following blood collection and centrifuged after venipuncture for 15 min and the supernatants were stored at -70°C until analysis. Vitamin A Testing was performed on healthy blood donors; the testing was performed by HPLC. Results: Our study showed that, There was no difference in the results were obtained with the serum (No Additive tube) and plasma tube in concentration of retinol 67.7 ug/dl and 73.8 ug/dl, but the results was 9.2 ug/dl obtained with the VACUETTE® Serum Sep Clot Activator tubes (gel tube). Conclusions: Tubes are drawn in a specific order to avoid the possibility of erroneous test results caused by carryover of an additive. If Serum tube-with or without clot activator or gel, it should be drawn as the first tube. We recommended that only serum and EDTA -plasma samples be used for Vitamin A determination by HPLC and cannot be carried out with all gel tubes.