Poly(ethylene glycols) (PEGs) are widely used water soluble and biocompatible polymers. PEGs are suitable as paracellular probes in biomembrane permeability studies because they are hydrophilic and various oligomers have defined molecular sizes. In previous studies corneal and conjunctival permeability for neutral PEGs has been measured, and the results were used to calculate the number and size of the cellular pores. In this study we have developed a high-performance liquid chromatographic-electrospray ionization-mass spectrometric method for analysis of neutral PEGs and positively changed amino PEGs simultaneously. The new method is fast, accurate, sensitive and specific for high throughput analysis. The method was used to evaluate the paracellular permeability of PEGs through a corneal epithelial cell culture. Paracellular pores are negatively charged and it was in our interest to characterize the interactions of positive charge and size of the molecules with the paracellular pores.