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Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass‐to‐charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility–mass spectrometry (DI‐IM‐MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se− (non‐secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2′‐fucosyllactose (2'‐FL) and lacto‐N‐fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI‐IM‐MS was also investigated. The standard addition method allowed the determination of the 2'‐FL despite the presence of other oligosaccharides, including 3‐fucosyllactose (3‐FL) isomer in breast milk. This proof‐of‐concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures. [ABSTRACT FROM AUTHOR]