Background Evidence shows that interprofessional collaboration (IPC) practice contributes to the quality of health care. However, there are limited instruments to assess IPC in providing primary care in the district health system (DHS) in Thailand. The aim of this study is to develop a valid and reliable instrument to assess the IPC competency of primary care team members in DHSs. Methods This study was designed as an exploratory mixed methods study. In the qualitative phase, 37 participants, including policymakers, practitioners, and academics with experience in primary care, were involved. Data were analysed using thematic analysis, and trustworthiness was verified by triangulation and peer debriefing. In the quantitative phase, content validity, exploratory factor analysis (EFA), confirmatory factor analysis (CFA), and reliability were conducted, and the final version of the questionnaire was evaluated with 497 participants. Results: The findings showed an I-CVI range of 0.86–1.00 and S-CVI/UA = 0.87 for 49 items with a 5-point Likert scale. EFA suggested six factors: 1) collaborative teamwork, 2) population- and community-centred care, 3) communication and mutual respect, 4) clarification of roles and responsibilities, 5) interprofessional reflection, and 6) interprofessional values and mixed skills. In the CFA results, the model fit indices were acceptable (CFI = 0.99, RMSEA = 0.049, SRMR = 0.043) or slightly less than the goodness-of-fit values (GFI = 0.84). All subscales showed acceptable Cronbach’s alpha values with a range of 0.86–0.94. Conclusions The developed IPC competency instrument was confirmed its validity and reliability that contributes to assessing the IPC competency of primary care teams in DHSs. This information provides evidence to support tailored intervention to promote the IPC competency of primary care team work to achieve a common goal.