Background The aim of the present study is to determine the influence of different enamel shades of various thickness on chroma and value of vita shade of dentin. Materials and methods Three enamel composite resin shades (Enamel white, grey and neutral) and one dentin shade (A2) from Amelogen Plus (Ultradent) was used. Ninety Enamel disk specimens of 0.5, 0.75 and 1 mm thickness and 10 mm in diameter for each shade and 90 dentin disk specimens of 2 mm in thickness and 10 mm in diameter was used for the study. The spectrophotometric values of the dentin shade with and without enamel specimens were recorded and the values were converted to CIE L*a*b values. Results Statistical analysis was done using Pearson correlation coefficients to verify the effect of thickness on Chroma and value, and the significance was evaluated by one-way ANOVA and Tukey post hoc test. Two way ANOVA and Tukey post hoc was done to verify the variation within the groups. Results revealed a significant positive correlation between thickness and chroma and a negative correlation between thickness and value. There was a statistically significant variation in between the groups. Conclusion All groups produced a significant increase in chroma with increase in thickness of enamel shade upto a thickness of 0.75 mm after which the behavior of each shade was erratic. Hence, the optimum thickness would be 0.75 mm. All groups produced a significant decrease in value with increase in thickness of enamel shade. Enamel white produced the greatest reduction in value, enamel neutral the least and enamel grey demonstrated an intermediate result. Clinical significance There is a need to have a knowledge of the effect on chroma and value when dentin is layered with different enamel shades, it is also important to understand the effect of these enamel shades at different thicknesses to better control the color and reproduce esthetic simulating natural teeth. How to cite this article Hajira NSWN, Mehta D, Ashwini P, Meena N, Usha HL. Influence of Different Enamel Shades and Thickness on Chroma and Value of Dentin Vita Shade: An in vitro Comparative Assessment Study. J Contemp Dent Pract 2015;16(4):304-309.