Background: To provide both preventive and rehabilitative conditions in a workplace, one must understand how employees experience work demands. Such an understanding can be obtained from each individual with valid and quality-assured questionnaires. The Work Environment Impact Questionnaire (WEIQ) is a new questionnaire for measuring employees’ self-perceived work ability in relation to their specific workplace environment. The purpose of this study was to assess the measurement properties in terms of construct validity of the WEIQ. Methods: A cross-sectional survey study was conducted with 288 respondents from three different workplaces involving assisted living personnel, vocational rehabilitation personnel and personnel at a research institute. The measurement properties of the WEIQ were assessed according to Rasch Measurement Theory (RMT), including assessment of item-to-sample targeting, threshold ordering, item fit statistics, unidimensionality and reliability. Results: Item fit, i.e., fit residuals, item characteristic curves (ICC) and chi square values, were all satisfactory, and no disordered thresholds were present after collapsing the lowest response categories. However, issues with local dependent (LD) item correlations was present in 7.6% cases, four items showed statistically significant differential item functioning (DIF), where 11% of the respondents had person fit residuals outside the recommended range of ± 2.5 and the t-test for unidimensionality did not meet the criterion of 5%. Scale-to-sample targeting and reliability (0.92) were good. LD could be resolved with testlets and at the same time maintaining fit and improving dimensionality, but then the reliability decreased to 0.82. Conclusions: This study provides an initial validation of the WEIQ to be used for assessing employees’ self-perceived work ability. Most measurement properties were acceptable, but further exploration of LD, DIF and unidimensionality in additional work settings and with larger sample sizes is warranted. Trial registration: Not applicable.