The two-phase extraction and spectrophotometric determination of polyoxyethylene non-ionic surfactants in water at trace levels is examined in detail by considering both monodisperse and polydisperse surfactants of the type RO(CH2CH2O)nH, where R = p-tert-nonylphenyl and n is the degree of polymerisation. Potassium picrate is used as a reagent for the polyoxyethylene chain and 1,2-dichloroethane as an extracting phase. Monodisperse surfactants with n from 4 to 15 were isolated by liquid - solid absorption chromatography. Their purity was checked by temperature-programmed gas - liquid chromatography. Their reactivity to the reagent is explained qualitatively by considering the equilibria involved in the extraction. Polydisperse surfactants with n̄ (number-average degree of polymerisation) ranging from 3.3 to 21.5 are also considered and compared with other polydisperse surfactants in which R = dodecyl. The concentration of these non-ionics in waters is conveniently expressed as potassium picrate active substances (PPAS). It can be referred to the standard synthetic monodisperse surfactant RO(CH2CH2O)6H, where R = dodecyl, which gives a spectrophotometric response acceptably near to that of the examined series of commercial surfactants.