Watermelon (Citrullus lanatus) is a fruit of high consumption in tropical countries, which contains a large number of essential micronutrients, vitamins, phenolic content and citrulline, co-producer of nitric oxide, precursor of arginine, efficient scavenger of hydroxyl radicals and a strong antioxidant that is present in greater proportion in its rind. The present work sought to determine the optimal conditions for obtaining citrulline-rich extract from the rind of watermelon (Citrullus lanatus) hybrid Santa amelia (Royal Sweet), using the centrifugation method. The response surface method (RSM) was used to optimize the process. The factors analyzed were acetic acid concentration (0.1-0.3M), extraction temperature (5 - 10°C) and extraction time (5 - 15 min) by the Box Behnken method with three replicates at the center point at 95% confidence and as output variable the citrulline yield quantified by spectrophotometry at 530 nm. Optimum conditions were found at 0.1 molar - 8.66 min - 5°C (p<0.05) with an optimum yield of 1.38% based on dry bark.