Background: There is evidence that physical activity plays an essential role in preventing illnesses during pregnancy. This study aimed to design and validation a theory-based physical activity assessment questionnaire for pregnant women (PA2Q-PW). Methods: In this exploratory psychometric study, a physical activity assessment questionnaire for Pregnant Women (PA2Q-PW) was designed. Face-to-face interviews were conducted to develop the initial items of PA2Q-PW. The findings from interviews were merged with the literature review. The face, construct, and content validity of PA2Q-PW was assessed by the participation of pregnant women, midwives, and health educationists. Impact Score (IS), Content Validity Index (CVI), and Content Validity Ratio (CVR) and of the items were calculated. Construct validity of PA2Q-PW was calculated through factor analysis. Intra-class correlation coefficient (ICC), Cronbach’s alpha, standard error of measurement (SEM), and composite reliability were calculated to assess the reliability of the findings by PA2Q-PW.Results: The highest score was seen in the Knowledge subscale of PA2Q-PW, and the value of Cronbach's alpha for the subscales was from 0.89 to 0.97, and the value of ICC was from 0.76 to 0.89. The correlation (CVI & CVR indices) between the experts was satisfactory in all items of PA2Q-PW. The minimum loading factor for varimax rotation in the knowledge subscale was 0.41, in attitude was 0.56, in nurtures was 0.38, and in enabling factor was 0.27. The fitness of the proposed model among those constructs was confirmed by Root Mean Square Error of Approximation (RMSEA) <0.08, Normed chi-square (x 2/ df) < 5.0, Comparative Fit Index (CFI) ≥ 0.90, Tucker- Lewis Index (TLI) ≥ 0.9.Conclusion: PA2Q-PW is the first standard tool based on the PEN-3 cultural model that researchers can utilize to gather data and conduct the desired education interventions to change physical activity behavior in pregnant women during pregnancy. Trial ID: ISRCTN13356179