Chemistry laboratories play an essential role in the education of undergraduate Science, Technology, Engineering, and Mathematics (STEM) and non-STEM students. The extent of student learning in any educational environment depends largely on the effectiveness of the instructors. In chemistry laboratories at large universities, the instructors of record are typically graduate or undergraduate teaching assistants (TAs). Despite the importance of their role in the education of undergraduate students, TAs' instructional practices have been largely understudied outside specific reform efforts. In this study, we developed a segmented observation protocol, the Laboratory Observation Protocol for Undergraduate STEM (LOPUS), in order to characterize TAs' instructional styles in a General Chemistry laboratory curriculum. LOPUS captures both students' and TAs' behaviors every 2 min as well as initiators of verbal interactions and the nature of these verbal interactions (e.g., data analysis, explanation of concepts). Analyses of 19 videos collected from 15 TAs resulted in the identification of four instructional styles: the waiters, the busy bees, the observers, and the guides on-the-side. We found that students' behaviors were independent of these styles and limited to performing the laboratory activities, initiating conversation with TAs, and asking TAs questions. Interestingly, students rather than TAs were initiators of most verbal interactions, regardless of TAs' instructional styles. Finally, we found that the nature of TA–student verbal interactions was related to the nature of the laboratory activity (e.g., only following step-by-step instructions versus carrying out extensive data analysis). Implications of these findings for future research investigations and TA training are discussed.