Purpose: The promotion of a sustainable, healthy and productive working life attracts more and more attention. Recently the Work Role Functioning Questionnaire (WRFQ) has been cross-culturally translated and adapted to Dutch. This questionnaire aims to measure the health-related work functioning of workers with health problems. The aim of this study is to evaluate the reliability, validity (including five new items) and responsiveness of the WRFQ 2.0 in the working population. Methods: A longitudinal study was conducted among workers. The reliability (internal consistency, test-retest reliability, measurement error), validity (structural validity-factor analysis, construct validity by means of hypotheses testing) and responsiveness of the WRFQ 2.0 were evaluated. Results: A total of N = 553 workers completed the survey. The final WRFQ 2.0 has four subscales and showed very good internal consistency, moderate test-retest reliability, good construct validity and moderate responsiveness in the working population. The WRFQ was able to distinguish between groups with different levels of mental health, physical health, fatigue and need for recovery. A moderate correlation was found between WRFQ and related constructs respectively work ability and work productivity. A weak relationship was found with general self-rated health, work engagement and work involvement. Conclusion: The WRFQ 2.0 is a reliable and valid instrument to measure health-related work functioning in the working population. Further validation in larger samples is recommended, especially for test-retest reliability, responsiveness and the questionnaire's ability to predict the future course of health-related work functioning.