The use of raffinose family oligosaccharides (RFOs) to control several diseases and stimulate immune systems has been studied. Rehmannia glutinosa is reported to contain RFOs; however, there are no reports on their content level in R. glutinosa. Furthermore, it is difficult to analyse RFOs using common reverse-phase chromatographic techniques due to their high polarity. To develop a rapid and reliable chromatographic method for the simultaneous quantitation of multiple RFOs in R. glutinosa. Chromatographic separation was performed by using hydrophilic interaction liquid chromatography coupled with an evaporative light scattering detector. For efficient ultrasound-assisted extraction of oligosaccharides, a central composite design was used. Response surface analysis estimated the highest-obtained yields of raffinose, stachyose and verbascose to be 58.31, 408.75 and 26.59 mg, respectively, from 1 g of R. glutinosa, while under optimal conditions, experimental yields per gram were 56.79, 391.42 and 27.69 mg, respectively. A rapid and reliable chromatographic method to simultaneously quantitate multiple RFOs in R. glutinosa was developed. Three RFOs in R. glutinosa were separated within 10 min using isocratic conditions. Quantitative analysis of RFOs showed that R. glutinosa may be a rich source of α-galacto-oligosaccharides, especially stachyose.