A simple and sensitive method for the simultaneous determination of chito-oligosaccharides (COSs) with degrees of polymerization (DPs) from 2 to 7 was developed and used for COS quantification in rat plasma. Samples were separated on a Waters XBridge Amide column (3.5 μm, 2.1 × 150 mm) by isometric elution with 10 mM aqueous ammonium acetate (pH = 9) in acetonitrile and 10 mM aqueous ammonium acetate (pH = 9) (v/v, 50 : 50) employing multiple reaction monitoring (MRM) detection. Analytes and internal standards (IS) were extracted from rat plasma by protein precipitation with acetonitrile. The assay was linear over a concentration range of 20-10 000 ng mL-1 for COS2-7. The intra-day and inter-day precision of the investigated components exhibited an RSD within 15%, and the accuracy (RE%) ranged from -7.3% to 7.6%. The extraction recoveries of the six constituents were determined to be between 82.5% and 94.3%. No significant matrix effects for COS2-7 were observed in rat plasma. COS in plasma remained stable for 24 h at room temperature (short-term), after freeze-thaw cycles, and 30 days in a -40 °C freezer. In comparison to reported COS quantitation methods, this method is simple, sensitive and cost-effective and could be used for the simultaneous quantitation of COS2-7. This method meets the Food and Drug Administration guidelines and had been successfully applied to the analysis of pharmacokinetic samples collected from rats.