Objective: This mixed-methods study aims to develop and validate a comprehensive hygiene tool from a One Health perspective to document the assessment of hygiene at the human-animal interface. Methods: This mixed-methods study consisted of two components: a narrative review of the development of tool, followed by validation. The tool development was conducted in two phases. Phase 1 was a literature review, while phase 2 focused on the development and validation. Results: Tool version 1 consisted of 37 questions to assess personal hygiene practices among 16 households with livestock. Face validity, content validity, and multi-rater kappa scores were used to validate tool version 1. The questions with kappa score exceeding 0.74 were retained for further test. A total of 29 questions were obtained and incorporated into tool version 2, which was subsequently employed to evaluate the personal hygiene practices among 62 households with stock. The median total scores for 16 households and 62 households were 10.5 (range: 6-16) and 16 (range: 10-22), respectively. Conclusions: Many studies have assessed hygiene practices among livestock farmers, yet none used a validated assessment tool. Thus, this validated tool will facilitate the measurement of hygiene in human-animal interactions.