Aim of the Study: This study was designed to study the effect of using different concentration and time of application of ozone gel on tooth whitening by measuring the change in color index of teeth. Materials and Methods: forty Rectangular enamel slabs were prepared from selected bovine Incisor teeth. The prepared specimens were divided into 2 main groups (20 specimens) according to concentration of ozone (95μg/ml at 1 ml3/sec and 190 μg/ml at 1 ml3 /sec) and each group subdivided into 2 subgroups (10 specimen each group) according to the time of application (eight hours daily for 7 or 14days). The ozone gel was applied to half of each prepared specimen according to assigned groups and the other half was left untreated to serve as a baseline control. At the end of treatment protocol both the control and treated sides of each group were assessed for color changes using spectrophotometer. Results: Increasing the color change by the high concentration of ozone gel in recommended time and in reduced time where ΔE were more than 3 in both times, while color changes were non significantly with low concentration of ozone where ΔE less than 2. Conclusion: Preparation of Ozone gas in gel form is a viable and promising method for preserving the Ozone gas and increasing its shelf life. The enamel color change was both concentration and time dependent.