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Dietary interventions have captured the attention of nutritionists due to their health-promoting aspects, in addition to medications. In this connection, supplementation of nutraceuticals is considered as a rational approach to alleviating various metabolic disorders. Among novel strategies, prebiotic-supplemented foods are an encouraging trend in addressing the issue. In the present investigation, prebiotic fructooligosaccharides (FOS) were extracted from garlic (Allium sativum L.) powder using ultrasound-assisted extraction (UAE). The response surface methodology (RSM) was used to optimize the independent sonication variables, i.e., extraction temperature (ET, 80, 90, and 100 °C), amplitude level (AL, 70, 80, and 90%) and sonication time (ST, 10, 15 and 20 min). The maximum FOS yield (6.23 ± 0.52%) was obtained at sonication conditions of ET (80 °C), AL (80%) and ST (10 min), while the minimum yield of FOS was obtained at high operating temperatures and time. The optimized FOS yield (7.19%) was obtained at ET (80 °C), AL (73%) and ST (15 min) after model validation. The influence of sonication parameters, i.e., ET, AL and ST, on FOS yield was evaluated by varying their coded levels from −1 to +1, respectively, for each independent variable. High-performance liquid chromatography with refractive index detector (HPLC-RID) detection and quantification indicated that sucrose was present in high amounts (2.06 ± 0.10 g/100 g) followed by fructose and glucose. Total FOS fractions which included nystose present in maximum concentration (526 ± 14.7 mg/100 g), followed by 1-kestose (428 ± 19.5 mg/100 g) and fructosylnystoses (195 ± 6.89 mg/100 g). Conclusively, garlic is a good source of potential prebiotics FOS and they can be extracted using optimized sonication parameters using ultrasound-assisted techniques with maximum yield percentage. [ABSTRACT FROM AUTHOR]