<p>A simple analytical assay based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and micro-solid phase extraction (μ-SPE) techniques was established for the quantification of polypropylene glycol 4&#xa0;K (PPG4K) polymers in cellular samples. After μ-SPE, the analytes were concentrated and separated on a reversed phase C₁₈ column. Collision induced dissociation (CID) was tested and the parent ions of PPG4K polymers were fragmented with a relatively high value of declustering potential (DP) in the ion source. Numerous fragment ions associated with the structure of PPG4K were generated in ion source at <i>m/z</i> 58<i>n</i> + 1 ([M + H]⁺), 58<i>n</i> + 18 ([M + NH₄]⁺), 58<i>n</i> + 23 ([M + Na]⁺) and 58<i>n</i> + 39 ([M + K]⁺), respectively. [M + NH₄]⁺ adducts were selected and further fragmented in the second quadrupole of the UHPLC-MS/MS system. Multiple reaction monitoring (MRM) transition at <i>m/z</i> 598.4 [M + NH₄]⁺ (58 × 10 + 18) → 175.0 was selected to determine PPG4K polymers in cellular samples owing to its excellent selectivity and higher sensitivity compared with the other MRM transitions. The developed analytical assay was successfully applied to the bioanalysis of PPG4K polymers in cellular samples. The lower limit of quantification (LLOQ) was 10&#xa0;ng/mL and the limit of detection (LOD) was 2.5&#xa0;ng/mL for PPG4K polymers. This study provides a novel analytical strategy for the determination of PPG4K polymers, which is coupled with μ-SPE and the selection of [M + NH₄]⁺ adduct strategy to enhance sensitivity.</p>