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The objective of this study was to develop an assessment instrument to measure the effects of a continuing education intervention on 3 domains in pharmacists' knowledge needed to provide pharmaceutical care for transgender patients: (1) foundations of gender-affirming care, (2) health disparities and the specific needs of transgender patients, and (3) hormone treatments for transgender patients. Multiple-choice questions were developed, and an initial item bank of 47 items was drafted. Item bank revision was conducted by content matter experts, while feedback from 8 practicing pharmacists was provided for face validity and further insights. A preliminary test, containing 42 items was administered to 64 pharmacists before and after a three-hour continuing education intervention. Cronbach's alpha coefficient yielded a value of 0.65 as a pre-test and 0.77 as a post-test. Items were less difficult to answer by participants after taking the three-hour continuing education, showing better discrimination among high and low performers in the instrument administration as post-test, as well as better correlation when comparing participants' performance in the overall score against item-level performance. Psychometric evidence supports further instrument examination, which can improve this tool to measure gains in pharmacists' knowledge related to the care of transgender patients.