The seeds of Tamarindus indica L., classified as bio-waste, are powerful sources of bioactive compounds, especially tannins. In order to use its full potential, extraction of such bioactive constituents should be done under the optimized conditions. In the frame of this paper, central-composite experimental design and RSM (response surface methodology) were applied in order to investigate the impact of the maceration parameters on target responses and to optimize extraction process. Extraction was performed under the different levels of extraction solvent (methanol), temperature, and solvent-to-sample ratio. Obtained extracts were evaluated in terms of total phenols, flavanols, and tannin yield and in vitro antioxidant activity (DPPH, FRAP, CUPRAC). Experimental results were fitted to a second-order polynomial model where regression analysis and analysis of variance were used to determine model fitness and optimal extraction conditions. Optimized extraction conditions determined by RSM were methanol concentration of 69.99%, extraction temperature of 23.38 °C, solvent-to-sample ratio of 1:20. Chemical characterization of the extract obtained under the optimized conditions was done by using HPLC-ESI-MS/MS technique. The study could provide a scientific baseline for designing novel functional products from T. indica seeds in further studies.