Infant formula provides a good source of fructooligosaccharides, which are considered to be functional ingredients of food due to their positive effects on gastrointestinal function. This study presents a simple and specific ultra-high performance liquid chromatography–tandem mass spectrometry method for the ultrasensitive determination of three fructooligosaccharides in infant formula. The analytes were extracted using acetonitrile-water solutions. The compounds were separated with an amide column by gradient elution using acetonitrile and water as mobile phases. The identification and quantification were achieved by using electrospray ionization tandem mass spectrometry in negative ion mode with multiple reaction monitoring. Accuracy, precision, limits of quantification, and limits of detection of the method were obtained. Under the optimal conditions, all calibration curves for fructooligosaccharides targets showed good linearity (R2 ≥ 0.998). The limits of detection and quantification were below 0.82 and 2.74 ng mL−1, respectively. The recoveries of the method ranged from 98.5% to 104.4%. To the knowledge of the authors, this is the first quantitative determination of fructooligosaccharides in infant formula by ultra-high performance liquid chromatography–tandem mass spectrometry. The method may be suitable for the determination of trace concentrations of fructooligosaccharides in other food.