Nonylphenols (NP) are ubiquitous in the environment and show toxic effects and estrogenic activity. According to the corresponding framework directive in the field of water quality, isomers of NP (including 4-n-NP and branched 4-NP) are classified as priority hazardous substances and are regulated as a group of chain and ring isomers with a maximum acceptable concentration of 2 μg/L in inland and other surface waters. This study presents a new sensitive and innovative screening approach for estrogen active NP based on high-performance thin-layer chromatography. NP were focused in a single target zone on thin-layer plates by planar solid phase extraction (pSPE) and detected by a planar yeast estrogen screen (pYES) on the basis of their estrogenic activity. The mean limits of detection and quantitation were 14 and 26 ng per zone, respectively. After liquid-liquid extraction of water samples with dichloromethane, the mean recovery was close to 100% (relative standard deviation of 21% or less), and estrogen active NP were detectable down to 1 μg/L. Thus, pSPE-pYES provides both the detection and the quantitation of estrogenic NP in surface waters at the maximum acceptable concentration. Application of the approach on extracts of surface waters showed the use of pSPE-pYES for environmental samples, and no complex and time-consuming clean-up of the extracts was required. Estrogenic NP were not detectable in any of the investigated surface waters by means of the screening approach presented.