The General, Organic, and Biological Chemistry Knowledge Assessment (GOB-CKA) is a multiple-choice instrument designed to assess students' understanding of the chemistry topics deemed important to clinical nursing practice. This manuscript describes the development process of the individual items along with a psychometric evaluation of the final version of the items and instrument. In developing items for the GOB-CKA, essential topics were identified through a series of expert interviews (with practicing nurses, nurse educators, and GOB chemistry instructors) and confirmed through a national survey. Individual items were tested in qualitative studies with students from the target population for clarity and wording. Data from pilot and beta studies were used to evaluate each item and narrow the total item count to 45. A psychometric analysis performed on data from the 45-item final version was used to provide evidence of validity and reliability. The final version of the instrument has a Cronbach's alpha value of 0.76. Feedback from an expert panel provided evidence of face and content validity. Convergent validity was estimated by comparing the results from the GOB-CKA with the General-Organic-Biochemistry Exam (Form 2007) of the American Chemical Society. Instructors who wish to use the GOB-CKA for teaching and research may contact the corresponding author for a copy of the instrument.