Chitooligosaccharides (COSs) have various physiological activities and broad application prospects; however, their pharmacokinetics and tissue distribution remain unclear. In this study, a sensitive and selective ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for determining chitobiose (COS 2) and chitotriose (COS 3) in rat serum and tissues was developed. This method was successfully validated based on FDA guidelines in terms of selectivity, calibration curves (lower limit of quantification was 0.002 µg/mL for COS 2 and 0.02 µg/mL for COS 3), precision (intra-day relative standard deviation of 0.04%-3.55% and inter-day relative standard deviation of 1.94%-11.63%), accuracy (intra-day relative error of - 1.81%-11.06% and inter-day relative error of - 9.41%-8.63%), matrix effects, recovery (97.10%-101.29%), stability, dilution integrity, and carry-over effects. Then, the method was successfully applied to the pharmacokinetics and tissue distribution study of COS 2 and COS 3 after intragastric and intravenous administration. After intragastric administration, COS 2 and COS 3 were rapidly absorbed, reached peak concentrations in the serum after approximately 0.45 h, and showed rapid elimination with clearances greater than 18.82 L/h/kg and half-lives lower than 6 h. The absolute oral bioavailability of COS 2 and COS 3 was 0.32%-0.52%. COS 2 and COS 3 were widely distributed in Wistar rat tissues and could penetrated the blood-brain barrier without tissue accumulation.