Background: The burden of mental ill health in working-age populations has prompted research on possible links between work-related factors and mental ill health. Long working hours and night shift work are some of the factors that have been studied in relation to the risk of developing mental ill health. Yet, previous studies have not generated conclusive evidence, and further studies of high quality are needed. Objective: This study aims to investigate the prospective association between working time arrangements and mental health in terms of psychotropic drug usage or psychiatric hospital treatment in the general working population of Denmark. Methods: Data on total weekly working hours in any job and night shift work from the Danish Labor Force Survey 2000-2013 will be linked to data from the Psychiatric Central Research Register (expected 2400 cases during 700,000 person years at risk) and National Prescription Registry (expected 17,400 cases during 600,000 person years at risk). Participants will be followed for up to 5 years. We will use Poisson regression to separately analyze incidence rates of redeemed prescriptions for psychotropic medicine and incidence rates of psychiatric hospital treatment due to mood disorders, anxiety disorders, or stress-related disorders as a function of weekly working hours and night shift work. The analyses will be controlled for sex, age, calendar time of the interview, and socioeconomic status. Results: This is a study protocol. Power calculations indicate that the study has sufficient statistical power to detect relatively small differences in risks and minor interactions (eg, ∼90% power to detect a rate ratio of 1.1 for psychoactive medication use). We expect the analyses to be completed by the end of 2020 and the results to be published in 2021. Conclusions: In this study protocol, all hypotheses and statistical models of the project have been completely defined before we link the exposure data to the outcome data. The results of the project will indicate to what extent and in what direction the national burden of mental ill health in Denmark has been influenced by long working hours and night shift work.