Knowledge of gender identity and sexual orientation is essential to build more inclusive, equitable and respectful societies, where each person can live authentically without facing discrimination or stigmatization. The research focused on designing and validating an instrument to measure knowledge of gender identity and sexual orientation in Chilean higher education students. For this task, rigorous criteria were followed in the design and validation process: theoretical review, expert judgment, cognitive interview, and exploratory and confirmatory factor analysis. The instrument was administered to a convenience sample of 401 students belonging to a higher education institution located in the Biobío region, Chile. The results revealed a latent structure with adequate fit to the data, composed of 15 items underlying the variables of gender identity and sexual orientation. Reliability was established at an Omega coefficient of .87 and a Cronbach's alpha of .86, indicating satisfactory internal consistency. The instrument is a tool that seeks to promote inclusion, equity and understanding, by identifying areas of ignorance or misunderstandings that could contribute to discriminatory attitudes or unintentional exclusions in the context of higher education. It also provides information for the design of future educational programs that promote inclusion, equity, and understanding.