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Reference materials are useful for the quality control of analytical procedures and to evaluate the performance of laboratories. There are few and expensive certified reference materials commercially available for vitamin C or ascorbic acid analysis in food matrices. In this study, the preparation and the suitability assessment of an orange juice in-house reference material (RM) for vitamin C analysis in fruits, juices and in fruit pulps is described. This RM was used for the development and full validation of an HPLC method. The results showed excellent linearity (r² = 0.9995), good accuracy (96.6–97.3%) and precision, as relative standard deviation, ranged from 0.70% to 3.67%. The in-house RM was homogenous and stable at storage conditions (−80 °C) during 12 months. According to our results, this in-house RM is an excellent tool to use in quality control and method verification purposes for vitamin C analysis of …