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Abstract This paper presents the development and validation of the 17-item mathematics Graduate Student Instructor Observation Protocol (GSIOP) at two universities. The development of this instrument attended to some unique needs of novice undergraduate mathematics instructors while building on an existing instrument that focused on classroom interactions particularly relevant for students’ development of conceptual understanding, called the Mathematical Classroom Observation Protocol for Practices (MCOP²). Instrument validation involved content input from mathematics education researchers and upper-level mathematics graduate student instructors at two universities, internal consistency analysis, interrater reliability analysis, and structure analyses via scree plot analysis and exploratory factor analysis. A Cronbach-Alpha level of 0.868 illustrated a viable level for internal consistency. Crosstabulation and correlations illustrate high level of interrater reliability for all but one item, and high levels across all subsections. Collaborating a scree plot with the exploratory factor analysis illustrated three critical groupings aligning with the factors from the MCOP²(student engagement and teacher facilitation) while adding a third factor, lesson design practices. Taken collectively, these results indicate that the GSIOP measures the degree to which instructors’ and students’ actions in undergraduate mathematics classrooms align with practices recommended by the Mathematical Association of America (MAA) using a three-factor structure of teacher facilitation, student engagement, and design practices.