Free oligosaccharides (OS) are a significant functional component of milk that are difficult to quantitate. A simple method for quantitative analysis of the major free OS in bovine milk is described. Following a defatting step, protein elimination was performed by ultrafiltration. OS were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyzer in negative mode. The method is sensitive [with a limit of detection (LOD) for all representative OS of <0.1 ng] and reproducible, enabling simultaneous quantification of 13 major OS within a single run. Application of this method to the quantification of major OS in 32 milk samples collected after three different feeding treatments allowed us to reveal the relative abundance of different OS species, the variation of the OS content between individual cows, and the correlations between some of the major OS.