p-Nonylphenol (NP) is a ubiquitous degradation product of nonylphenol polyethoxylate (NPE) surfactants and has been reported to be an endocrine disrupter. It is composed of numerous structural isomers resulting from the various branching patterns of the C-9 group. Twenty-two isomers in a technical mix of NP have been identified with high-resolution capillary GC-MS. In most HPLC analyses, nonylphenol elutes as a single, broad peak. In the method described here, HPLC using a graphite carbon column resulted in the resolution of a technical mixture of NP into 12 peaks or groups of isomers. This method was also applied to select NPEs with one to 10 ethoxy units with similar results. Separation was achieved by gradient elution with 1% acetic acid in water and acetonitrile. Elution of individual isomers 4-butylphenol and 4-propylphenol under the same gradient conditions indicate that increased branching of an alkyl group results in shorter retention times than for the less substituted alkyl groups. This method can be used to fractionate NP based on structure and assess the potential for different isomers (or groups of structurally similar isomers) to act as endocrine disrupters.