Objective: To investigate the effect of dehydration time on tooth colour measurement in vitro using colourimetry. Methods: Ten extracted human maxillary central incisors were used. L*a*b* values at the centre of the labial surface of each tooth were measured using a colourimeter. The first measurement was performed after removal of excess water. The second and third measurements were performed after the teeth had dried at room temperature for 2 h and 4 h, respectively. The colour differences (ΔE) between dehydration time points were calculated. Repeated oneway ANOVA was performed for colour values at different dehydration time points. Pairwise comparison of group means (Student t test) was used to examine the differences between ΔE and 1.5ΔE (α = 0.05). Results: There were statistically significant changes in the L* values after 2 h and 4 h dehydration as well as between each ΔE and 1.5ΔE. Neither 2 h nor 4h dehydration resulted in change of a* value or b* value. Conclusion: Dehydration time affected colour measurement using a colourimeter; the teeth become lighter after dehydration for 2 h or longer. Keywords: colour measurement, dehydration, tooth, colour difference, colourimeter