Aim: The aim of the study was to evaluate the anxiety levels in children while using rubber dam and OptraDam isolation techniques. Materials and methods: This study was a crossover trial conducted on 27 selected 6-12-year-old children. The procedure of placement of either of the isolation techniques was told and demonstrated using audiovisual aid. The sequence of the proceedings on each child (rubber dam or OptraDam) was determined randomly using toss of coin. Second demonstration was carried out 7 days after the first demonstration. The anxiety experienced was recorded using Venham's anxiety scale at two time points-after verbal explanation and after the audiovisual demonstration. The study also objectively assessed the anxiety by measuring the salivary malondialdehyde (MDA) levels of two patients. Results: When mean values of Venham's anxiety scores after verbal explanation and after audiovisual demonstration were compared for each of the two techniques using paired Student's t test, there was statistically significant decrease in the anxiety score following audiovisual demonstration in both the techniques. When the scores between two groups after verbal explanation and after audiovisual demonstration were compared using repeated measures of analysis of variance (ANOVA), the reported anxiety scores were significantly lesser for the OptraDam technique (p = 0.000). Conclusion: Audiovisual demonstration reduced the anxiety of children when compared to verbal explanation for both isolation techniques. OptraDam isolation was found to be less anxiety generating in children compared to rubber dam isolation. Clinical significance: When using modern adhesive techniques, a good isolation of the working field is an important requirement for better prognosis. OptraDam being the latest addition to the rubber dam family, if found to be more children friendly can solve majority of the problems related to isolation in pediatric dentistry. How to cite this article: S Mahima, YM Karuna, Shenoy R, et al. Evaluation of Anxiety Levels in Children while using Rubber Dam and OptraDam Isolation Techniques. Int J Clin Pediatr Dent 2023;16(2):287-291.