Medical assistants—key professionals supporting physician practices—have not been studied with regard to burnout and professional fulfillment, which may affect other healthcare professionals. This study examined the factors associated with burnout among medical assistants in an academic healthcare organization while validating the use of a tool previously used to assess burnout in physicians. Using portions of the Professional Fulfillment Index (PFI) and questions designed for this mixed methods study, medical assistants employed across Stanford Health Care were surveyed. The authors assessed demographic characteristics and the impact of control, organizational culture, team knowledge, self-efficacy, and professional fulfillment/meaningfulness on burnout. Of the 505 eligible participants, 261 (52%) completed the survey; 76% were women. The study validated the PFI for use with this population and validated three additional scales. Burnout was found to be low among medical assistants (M = 2.32); professional fulfillment/meaningfulness of work was found to be high (M = 4.08). Organizational culture, professional fulfillment, and self-efficacy were found to be predictors of burnout (R2 = 0.438), with negative perceptions of organizational culture as the strongest predictor of burnout among medical assistants (β = –0.34). These results indicate that a survey tool is useful in understanding components of burnout and professional fulfillment in this population. Although limited to one site, this study could be replicated in other organizations.