The aim of this study was to determine the reliability and validity of a new test food for the clinical assessment of masticatory performance. The test food had two overlapping acidic/basic halves. Ten dentate subjects chewed one unit for 10-100 cycles. One subject chewed ten units for the same number of cycles. Differences in the L*, a* and b* colour axes were determined before and after chewing by ANOVA. Colour guides were created based on the values of these axes and matched with the number of cycles. The reliability of the guides was evaluated using 30 images, where three examiners indicated the number of cycles in which the colour closest to that of a chewed material was found. The data were contrasted with the real values of the guides to determine the validity (Kappa coefficients). The equivalence of the guides with the median particle size (X50) was determined using equidimensional curves. The test food progressively changed from green to pink during chewing. As the number of cycles increased, the a* values increased and the b* values decreased (p < 0.05). Overall, the guides showed a Kappa value >0.8 for the intra-examiner and inter-examiner reliability and the validity comparisons. The a* and X50 values were inversely proportional to each other, and the b* values showed a direct relationship with the X50 values. The L* values did not show correspondence. The new test food showed high reliability and validity for the assessment of masticatory performance through clinical colour guides matched with the number of cycles and X50.