Most research assessing human immunodeficiency virus (HIV) anxiety relies on single-item measures or psychometric measures that are outdated in terms of concepts and language. There is a critical need for a robust, reliable, and contemporary measure to identify populations at risk of avoiding HIV testing, treatment, and prevention, thereby supporting global HIV eradication goals. Focus groups informed the initial development of the HIV Anxiety Scale (HAS), revised through expert feedback. The factor structure was assessed in two studies. In Study 1, an Exploratory Factor Analysis (EFA) was conducted with 251 participants. In Study 2, a Confirmatory Factor Analysis (CFA) with 200 participants was performed alongside validity, internal consistency, and measurement invariance assessments. Studies 1 and 2 elicited a 3-factor model, resulting in a 16-item measure with the following subscales: Psychosocial Implications of HIV, Lifestyle Implications of HIV, and HIV Testing Anxiety. The HAS demonstrated a good factor structure, acceptable validity and excellent internal consistency across diverse groups in Study 2. The HAS provides a contemporary, robust measure of HIV anxiety, addressing limitations of previous tools and contributing to efforts to identify and support populations at risk of HIV avoidance behaviours. We recommend that future research continue to validate and test this new measure, but it offers a standardised tool to inform targeted interventions for HIV testing, prevention, and treatment.