A rapid and precise HPLC method with evaporative light scattering detection (ELSD) for the separation and quantification of polyethyleneglycol 2000 (PEG 2000), polyethyleneglycol 6000 (PEG 6000) and poly(methyl vinyl ether-co-maleic anhydride) (Gantrez) in a nanosized pharmaceutical formulation has been developed. Separation was carried out on a PL aquagel-OH 30,8μm column (300mm×7.5mm), in a gradient elution with methanol–water as mobile phase at a flow rate of 1ml/min. Quantification was determined in supernatants of PEGylated nanoparticles and the quantification limits were found to be 0.075mg/ml for polyethyleneglycols and 0.25mg/ml for Gantrez. The precision did not exceed 8% and accuracy range for PEGs (−11.50 and 10.61%) and Gantrez (−12.18 and 14.81%) were always within the acceptable limits. The amount of polyethyleneglycol associated to nanoparticles was also calculated by a Nuclear Magnetic Resonance Method ( 1 H NMR). Likely, for both PEGs, a good relationship between both techniques was found. In summary, the developed HPLC technique provides an alternative for the routine and rapid analysis of PEGs and Gantrez in nanoparticle formulations.