An increasing average herd size and complexity in farm structures call for a higher level of biosecurity. It can reduce the risk of introducing and establishing pathogens with multiple-pathway and indirect spread mechanisms, such as Salmonella Dublin, a pathogen with an increasing occurrence in dairy cattle farms across different countries and continents. Therefore, this study aimed to use existing knowledge to develop a framework with a supporting tool allowing for a time-efficient, yet comprehensive, assessment of biosecurity measures that can help prevent the introduction and establishment of S. Dublin in dairy herds. Based on the literature review, a seven-step biosecurity assessment framework was developed and evaluated in collaboration with biosecurity experts. The resulting framework includes a weighted semi-quantitative assessment method with a scoring guide in an electronic supporting tool for 12 biosecurity sections assessed through on-farm observations and farmer interviews. The framework and tool provide a novel approach to comprehensively assess the overall (mainly external) on-farm biosecurity level by a trained biosecurity assessor. They can be used for systematic data collection in epidemiological studies on risk factors for the introduction and establishment of S. Dublin in dairy farms. Preliminary interrater reliability testing indicated moderate reliability between assessors with varying biosecurity skills.