The dust level is included in the animal welfare legislation of the European Union, implying assessment of dust levels during veterinary welfare inspections. This study aimed to develop a valid and feasible method for measuring dust levels in poultry barns. Dust levels were assessed in 11 layer barns using six methods: light scattering measurement, the dust sheet test with durations of 1 h and 2–3 h, respectively, visibility assessment, deposition assessment, and a tape test. As a reference, gravimetric measurements were obtained – a method known to be accurate but unsuitable for veterinary inspection. The dust sheet test 2–3 h showed the highest correlation with the reference method with the data points scattered closely around the regression line and the slope being highly significant (p = 0.00003). In addition, the dust sheet test 2–3 h had the highest adjusted R2 (0.9192) and the lowest RMSE (0.3553), indicating a high capability of predicting the true concentration value of dust in layer barns. Thus, the dust sheet test with a test duration of 2–3 h is a valid method for assessing dust levels. A major challenge is the test duration as 2–3 h is longer than most veterinary inspections. Nevertheless, results showed that potentially, with some modifications to the scoring scale, the dust sheet test may be reduced to 1 h without losing validity.