Chemistry concepts are embedded in several contexts that are related to the future professional roles of pre-medical students. In the current academic landscape of pre-medical programs, an objective measurement of prior knowledge of chemistry concepts is necessary to promote students’ academic success. To address this issue, this study aimed to develop and validate the Prior Knowledge of Chemistry Concepts Test. This multiple-choice research instrument is designed to measure pre-medical students’ knowledge of essential chemistry concepts from pre-requisite chemistry courses in pre-medical programs in the Philippines. The final item pool, consisting of 26 general chemistry concepts and 21 general organic chemistry concepts was administered to 470 college students from four tertiary institutions in the Philippines. Item content validity indices (I-CVI) and kappa statistics guided initial revisions, while dichotomous Rasch analysis was utilized to determine the psychometric properties of the research instrument. The analysis provided evidence of adequate person and item reliability, item fit, local independence, and unidimensionality, although some items may be omitted in subsequent iterations. The application of the research instrument to assessment and evaluation was elaborated using the student ability and item difficulty logits along the person-item map. Suggestions for the improvement of the research instrument were also discussed.