In the present work solid-phase extraction protocol based on C18 tubes and organic water washing solvents as well as isocratic HPLC procedure focused on quantification of free steroids and related low-molecular-mass endocrine disrupting compounds (EDCs), characterized by different polarity varied from estetrol to progesterone, were studied. Described separation method involves temperature-dependent inclusion chromatography with mobile phase modified with beta-cyclodextrin. Using such analytical approach the environmental samples derived from Baltic Sea, selected lakes and rivers of the Middle Pomerania in northern part of Poland as well as untreated and treated sewage water from municipal sewage treatment plant near Koszalin were analyzed. Moreover, some preliminary data concerning estriol, testosterone and equilin biodegradation involving activated sludge material were reported. Cluster and principal components analysis of the acquired data sets confirms a high separation and quantification throughput of the solid-phase extraction and isocratic HPLC protocols presented. The method can be useful for simple and rapid classification of the environmental samples characterized by different sources of EDCs loading. The results of this work extend the utility of temperature-dependent inclusion chromatography as an inexpensive, efficient and accurate analytical tool appropriate for characterisation and quantification of complex environmental samples.