Poly(ethylene glycols) (PEG) are the major metabolites of non-ionic surfactants (NS). Chains of PEG may be shortened due to further biodegradation. The separate determination of short- and long-chained PEG (SC- and LC-PEG) may be useful to control the biodegradation of NS and PEG. A scheme for the separation of a whole mixture of PEG into SC- and LC-fractions and their subsequent determination is developed. A sequential extraction of a water sample with n-hexane and ethyl acetate is used to separate NS and other interferents as well as dichloromethane and chloroform for the separation of PEG into LC- and SC-fractions. The indirect tensammetric technique (ITT) is used for the final quantification of PEG into separated fractions as well as for the control of every separation step. The boundary between fractions is not sharp due to the similarity of the properties of neighbouring homologues which may be approximately 15 oxyethylene subunits. The developed scheme is successfully used for the separation into fractions of PEG from river water and raw and treated sewage. An initial separation of PEG from these samples by extraction with chloroform is carried out.