A deconstruction of the existing concept of internalized homophobia demonstrated the need for a new understanding of this experience and a different means of operationalizing the construct in men who have sex with men. This study introduces a new unified theory of sexual prejudice and a valid and reliable new psychometric tool to assess the concept of internalized sexual prejudice (ISP). A sample of 125 men who visited websites popular among men who have sex with men (MSM) were recruited to complete a survey that assessed ISP. Instrument development procedures, including factor, reliability, and validity analyses were conducted to refine the new ISP measure. Participants also completed the Center for Epidemiologic Studies Depression scale (CESD), the Rosenberg Self esteem scale (RSE), the Reactions to Homosexuality Scale (RHS), and the Sexual, and Substance Use Risk Behavior (SSRUB) questionnaires to aid in validation. The results indicated a valid construct of internalized sexual prejudice consisting of the 5 domains supported by theory. Validity was substantiated by the ability of the tool to predict certain sexual and substance use behaviors. Practitioners have a new assessment tool useful in continuing treatment of MSM. The behavioral HIV intervention campaigns in the MSM population can modify their social marketing campaigns to account for the experiences of ISP among MSM.