Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched. Traditionally, HMO profiling was performed using HPLC with fluorescence or UV detection. By using porous graphitic carbon liquid chromatography MS, it is now possible to separate and identify most of the isomers, facilitating linkage-specific analysis. Matrix-assisted laser desorption ionization time-of-flight analysis allows fast profiling, but does not allow isomer separation. Novel MS fragmentation techniques have facilitated structural characterization of HMO that are present at lower concentrations. These techniques now facilitate more accurate studies of HMO consumption as well as Lewis blood group determinations.