<b>Background and Objective:</b> An increase in the consumption of robusta coffee resulted in an increase in waste from coffee, one of the coffee wastes, namely coffee bean skins or cascara. Robusta coffee cascara contains 1-1.3 g of caffeine which causes side effects, such as insomnia and seizures etc. So this research aims to reduce the caffeine content in cascara by using <i>Bacillus subtilis</i>. Using optimum conditions and capabilities. <b>Materials and Methods:</b> The experiment was conducted from May to August, 2022 in the Pharmacy Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Pakuan, Indonesia. Before optimizing, cascara was extracted using the ultrasonic assisted extraction (UAE) method, validated by the High-Performance Liquid Chromatography (HPLC) method to determine caffeine content and a paired sample t-test was performed using Statistical Package for the Social Sciences (SPSS). <b>Results:</b> It showed that in validating the HPLC method, the wavelength of caffeine in cascara was 272 nm. The mobile phase was a mixture of methanol-water (adjust orthophosphate). The pH (2.4) (45:55), obtained the optimum decaffeination conditions at the concentration of bacteria <i>Bacillus subtilis</i> 6% and a long incubation time of 24 hrs resulted in a decrease in caffeine content of 51.3843±0.2503%. <b>Conclusion:</b> The results of the paired sample t-test indicate that the concentration of bacteria <i>Bacillus subtilis</i> and incubation time significantly influence caffeine levels.
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