Students majoring in biology typically take a semi-prescribed series of courses aimed at helping them master central concepts, develop practical competencies, and cultivate higher-order cognitive skills. We have developed a molecular biology capstone assessment to gauge student understanding of core molecular biology concepts and their ability to apply these concepts to novel scenarios. Targeted at senior-level students, this assessment utilizes a unique multiple true-false format where each question consists of a narrative stem followed by four true-false statements. Each question was developed with extensive faculty and student input, including content-validation through faculty feedback and response-validation through student interviews. Results from an online pilot administration of this assessment indicate that advanced students exhibit a wide range of achievement levels across different conceptual areas. Once complete, this assessment will provide a valuable tool for departments to pinpoint areas of conceptual difficulty and guide subsequent curricular reform.