The novel O-glycan relative quantitation method is based on a simple, optimized sample preparation approach that can achieve nonreductive release and stable isotope labeling of glycoprotein O-glycans in one pot and sample purification using just C18 SPE column. It features a permanent 10-Da mass difference between the light and heavy form derivatives of each O-glycan, allowing O-glycan relative quantitation of various biological samples with satisfactory quantitation linearity, reproducibility and accuracy by MS, MS/MS and on-line LC-MS