¹Research and Development, Deva Holding A.Ş., Çerkezköy-2, Atatürk Mh. Fatih Blv. No: 26 Kapaklı, Tekirdağ, Turkey DOI : 10.12991/jrp.2018.89 The water-soluble vitamins are gaining rapid importance not only for human health but also being widely recommended for animal health care because of theirs wide spectrum of activities ranging from deficiency, pathogenic condition to nutritional supplements. A plethora of articles are published for simultaneous evaluation of B-Complex Vitamins, but most of their methods are not selective and time consuming which involve pre-treatment of sample due to complexities involved in the placebo matrix. In this study, we propose a simple, specific, precise, accurate and linear method to separate vitamin B1 (thiamine), vitamin B2 (riboflavin sodium phosphate), vitamin B3 (niacin amide), vitamin B6 (pyridoxine) and vitamin C (ascorbic acid) by using Zorbax XDB C18 column (250 mm x 4.6 mm i.d., 5μm). The flow rate was kept as 1.3 mL/min and monitored at 270 nm with ambient temperature. Effective separations and quantification were obtained in 30 minutes with 10 μl injection volume. The current reverse phase gradient method resulted in significant resolution between interested vitamins and placebo matrix. Analytical method development, followed by analytical method validation was accomplished according to International Conference of Harmonization guidelines (ICH). Statistical evaluation of validation parameters proved that the results were highly specific, precise, accurate, robust and linear over a wide concentration range. This method has been validated and found to be applicable in routine analysis of vitamin B1, vitamin B2, vitamin B3, vitamin B6 and vitamin C in oral powder for veterinary consumption. Keywords : RP-HPLC; simultaneous determination; vitamin C, B1, B2, B3, B6; water-soluble vitamins; veterinary