Abstract: Polymer-drug conjugates have shown great potential in the field of drug delivery, but existing systemic pharmacokinetic data are difficult to clarify their pharmacokinetic behavior at the cellular level. In this study, multi-loaded pegylated irinotecan conjugate (PEG-irinotecan₃) was used as a model drug to build a research idea based on the interaction of polymorphic components to analyze its cellular pharmacokinetics. An HPLC-MS/MS analysis method based on in-source collision-induced dissociation (IS-CID) technology was established to achieve accurate quantitative analysis of PEG-irinotecan₃ and its intracellular polymorphic components, and comprehensively clarify its intracellular uptake, efflux and subcellular distribution characteristics. Uptake studies have shown the presence of polymorphic components in cells such as bonded drugs (PEG-irinotecan_{x (x = 3, 2, 1)}), release material (PEG20K), free drug (irinotecan) and active metabolite (SN-38). Subcellular distribution studies have shown that bonded drugs and release materials are mainly distributed in the cytoplasm, while irinotecan is mainly distributed in the cytoplasm and nucleus. This study expands the cellular pharmacokinetic research system of polymer-drug conjugates, and the key information obtained is of great value in guiding drug design, clarifying intracellular pharmacokinetic behavior, and promoting clinical transformation.