In the present work we have developed an analytical methodology for the determination of nonylphenol (NP) and nonylphenol mono- and di-ethoxylates (NP1EO and NP2EO) in water samples. The applicability of this methodology was proved by means of the analysis of environmentally relevant aqueous samples from Buenos Aires. This constitutes a starting point for a rigorous assessment of the incidence of NPnEO surfactants in Argentina, as only very few, qualitative or semi-quantitative data on the occurrence of these compounds in local systems were available up to this time. Enrichment of the analytes was carried out by solid-phase extraction on a C-18 sorbent, followed by elution with ethyl acetate. Normal-phase high performance liquid chromatography on an amino-silica column and fluorescence detection at excitation-emission wavelengths of 230–300 nm were employed for separation and quantification of the analytes. Confirmation of peak assignment in selected real samples was performed by off-line coupling HPLC with GC-MS analysis. A non-polar GC capillary column was used, and a characteristic peak pattern was obtained for the alkyl chain isomers of each ethoxylated homologue and NP. GC-MS analyses yielded in all cases purity levels higher than 80% for the HPLC collected fractions. The elevated concentrations found for the estrogenic metabolites of NPnEO are in accordance with an unrestricted use of this class of non-ionic surfactants in the country.