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The investigation of the pharmacokinetic behavior of PEG is important for revealing the toxicity and efficiency of PEG\u2010related Nano\u2010drug delivery systems. A high through\u2010put and selective ultra\u2010high\u2010performance liquid chromatography with tandem mass spectrometry (UHPLC\u2010MS\/MS) method coupled with collision\u2010induced dissociation (CID) in source technique was developed and validated to determine PEG1K polymers in cellular samples in this study. The countless precursor ions of PEG1K are dissociated in the source to generate numerous product ions which have different numbers of subunits. The transition of [M+H]<jats:sup>+<\/jats:sup> precursor ions \u2192 product ions at <jats:italic>m\/z<\/jats:italic> 177.1 (four subunits)\u219289.1 (two subunits) was selected to determine PEG1K due to its high sensitivity. The UHPLC\u2010MS\/MS method coupled with CID in the source showed good linearity over the range of 0.1\u201310\u00a0\u03bcg\/mL. 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