Instruments for assessing secondary students' conceptual understanding of core concepts in biology are needed by educational practitioners and researchers alike. Most instruments available for secondary biology (years 9 to 12) focus only on highly specific biological concepts instead of multiple core concepts. This study describes the development of a 25-item instrument designed to fill this gap, the High School Biology Concept Inventory (HS-BCI). The HS-BCI not only assesses student knowledge of key biological concepts but also alternative conceptions. Using Rasch theory, the initial instrument was constructed from a pool of 61 instrument items using test results from 1015 students. The final 25-item instrument was validated with 1955 students. The results provide reliability and validity evidence for the HS-BCI. The findings suggest that it can be utilized to assess both conceptual knowledge and alternative conceptions.