Background: Sitting time is an emerging health risk, and many working adults spend large amounts of time sitting each day. It is important to have reliable and accurate measurement tools to assess sitting time in different contexts. Objective: To validate the Workforce Sitting Questionnaire (WSQ), an adapted measure of total and domain-specific sitting time based on work and non-workdays for use in working adults. Methods: A convenience sample (N=95, 63.2% women) was recruited from two workplaces and by word-of-mouth in Sydney, Australia. Participants completed the WSQ, which asked about sitting time (1) while travelling to and from places; (2) while at work; (3) while watching TV; (4) while using a computer at home; and (5) while doing other leisure activities on work and non-workdays on two occasions, 7 days apart. Participants also wore an accelerometer for the 7 days between test and retest. They recorded the times they wore the accelerometer, the days they worked and their work times in a logbook. Analyses determined test-retest reliability with intraclass correlation coefficients (ICCs) and assessed criterion validity against accelerometers using Spearman's r and Bland-Altman plots. Results: Measuring total sitting time based on a workday, non-workday and on average had fair to excellent test-retest reliability (ICC=0.46-0.90) and had sufficient criterion validity against accelerometry in women (r=0.22-0.46) and men (r=0.18-0.29). Measuring domain-specific sitting at work on a workday was also reliable (ICC=0.63) and valid (r=0.45). Conclusions: The WSQ has acceptable measurement properties for measuring sitting time at work on a workday and for assessing total sitting time based on work and non-workdays. This questionnaire would be suitable for use in research investigating the relationships between sitting time and health in working populations.