PEGylation is practically one of most important modifications of drugs including small molecules, peptides and proteins, which has been proven to dramatically improve physicochemical properties and pharmacokinetic behavior of the PEGylated drugs. However, it is a challenge currently to quantitatively analyze PEG and PEGylated drugs by various analytical methods, even mass spectrometry because of multiple parent ion distribution of PEG caused by its polydispersity of molecular weight. Here we developed a robust method with MS/MSALL technique using electrospray ionization (ESI) source coupled high resolution Quadrupole Time-of-Flight (Q-TOF) mass spectrometry for the quantification of PEG2K-Paclitaxel (PEG-PTX) and its two metabolites, PEG and Paclitaxel (PTX). The analysis was performed on a 300SB-C18 column with acetonitrile and 0.1% formic acid as the mobile phase. Samples were simply prepared by protein precipitation in a small quantity of plasma (50 μL). Calibration curve was linear within the range of 50.0–4000 ng/mL for PEG and PEG-PTX and 1.0–1000 ng/mL for PTX. The intra- and inter-day precisions were 3.2–6.9% and 3.1–6.9% for PEG, 4.1–7.8% and 4.0–9.9% for PEG-PTX, and 3.3–4.8% and 3.1–6.9% for PTX, respectively. The recoveries were greater than 90% with low matrix effects. Afterwards, the newly developed method was successfully applied to support a preclinical pharmacokinetic study in six rats after single intravenous injection of PEG-PTX (51.7 mg/kg).