Abstract Background Retinol-binding protein (RBP) is a liver-synthesized transport protein responsible for delivering retinol (vitamin A) from the liver to target tissues. It serves as a sensitive biomarker for nutritional status, with decreased serum and plasma levels indicating undernutrition and vitamin A deficiency. Conversely, elevated RBP levels are associated with conditions such as renal failure and type-2 diabetes, highlighting its clinical relevance in both nutritional and metabolic health assessments. This study aimed to establish the reference range for RBP using turbidimetric RBP assay from Gentian AS on the Beckman Coulter DxC 700 AU analyzer. In addition, potential differences between fresh and frozen samples and across different biological matrixes have been investigated. Methods A total of 121 samples from healthy volunteers ranging from age 16 to 69 years, including both fresh and frozen lithium-heparin plasma, were analyzed on Beckman Coulter DxC 700 AU analyzer. A Box-Cox transformation was applied, and the reference range was determined using normal quantile analyzis. Results The established reference range for RBP was found to span from 24 to 62 mg/L with a 90% confidence interval. The results exceeded previously reported reference range (30–60 mg/L) (Tietz Textbook of Clinical chemistry and molecular Diagnostics, 4th ed, 2006), emphasizing the importance of assay-specific validation for accurate clinical interpretation. No significant difference was found in analyzis of fresh and frozen samples. The reference range for males and females were slightly different (males, n=54: 22-61 mg/L and females, n=67: 19-57 mg/L). Further analyzis will be done to confirm the difference and evaluate its significance. Additionally, no significant variation was observed between serum, lithium-heparin plasma, and EDTA plasma matrixes, indicating strong consistency and reproducibility of results across sample types. Conclusion This study provides reference range for Gentian RBP assay and highlights the reliability and robustness of the assay across sample types and storage conditions. The findings underscore the necessity for manufacturers and/or laboratories to establish assay-specific reference ranges to ensure accurate clinical assessments.