Summary: Weight bias among healthcare professionals and healthcare students is pervasive and encompasses negative attitudes, beliefs, and discrimination toward patients with obesity. Weight bias in healthcare has unique negative effects, including less information shared with patients and healthcare avoidance. While several measures exist that assess weight bias, few measures have been designed specifically to assess weight bias in healthcare. The current, multiphase study was designed to address this gap via development and validation of such an instrument. To allow for statistical control of overly favorable responding, the proposed instrument includes a social desirability subscale (also designed specifically for use among healthcare professionals). Eleven focus groups were conducted with current healthcare professionals, healthcare students, and patients who have experienced weight bias to better understand weight bias in healthcare and inform item development. Ten different major and minor themes were identified. Four themes emerged as major themes across all types of focus groups: Weight Assumed to be the Cause of All Problems, Timing and Frequency of Weight-Related Discussions, Providers’ Assumptions/Stereotypes, and Providers’ Communication/Interactions Show Bias. Instrument items were created based on major and minor themes identified from the focus groups. Participants (N = 112) were recruited across two pilot phases to conduct initial item analyses and refine the instrument. An exploratory and confirmatory factor analysis of the refined instrument was conducted with 178 participants. The final instrument, titled the Weight Bias in Healthcare Scale, allows for more accurate assessment of weight bias specific to the healthcare setting. This instrument can be used to determine the efficacy of bias reduction interventions and can be incorporated into training programs and hospital settings to determine if and when such interventions are necessary.