The purpose of this investigation was to explore changes in undergraduate and graduate teaching assistants' (TAs') content knowledge and beliefs about teaching within the context of an inquiry-based laboratory course. TAs received professional development (PD), which was informed by the TA training literature base and was designed for TAs implementing a guided inquiry approach to general chemistry laboratory instruction. TAs engaged in ~20 h of presemester PD and ~30 h of weekly follow-up PD during the semester. The study utilized a multiple-methods approach within a social constructivist framework to assess changes in the TAs. Participants included eight graduate TAs and five undergraduate TAs. Data collection included TA pre-PD, post-PD, and semester-end surveys and two interviews of a subset of participants. The quantitative data were analyzed using descriptive and nonparametric statistics, and the qualitative data were analyzed using systematic data analysis. The results indicate that TAs' content knowledge significantly improved following the PD (mean = 80.22, standard deviation = 11.80) (Z = -2.346, p = 0.019) and was maintained over the semester. Following PD, the TAs shifted their beliefs to be more aligned with inquiry-based instruction. The results of this investigation suggest that TA previous experience and teaching students in an inquiry-based lab may influence TAs' beliefs. Future research will focus on examining the impact of TAs on student outcomes within a guided inquiry approach to general chemistry laboratory instruction.