We present a novel tool for detecting and monitoring photosystem II (PSII) inhibitors, using the freshwater alga Desmodesmus subspicatus, in environmental samples fractionated by high-performance thin-layer chromatography (HPTLC). After chromatographic separation of a sample on a HPTLC plate, the algal suspension is sprayed homogeneously on the plate, and PSII-inhibition by specific sample components is detected based on changes in fluorescence yield, viewed by a maxi Imaging-Pulse-Amplitude-Modulation fluorometer. Dose-dependent responses to the PSII-inhibitor herbicides atrazine and diuron, frequently detected in water bodies, are demonstrated without and with chromatographic separation. The limits of quantification for atrazine and diuron with chromatographic separation were 1.94 ng and 99 pg, respectively, allowing the detection of environmentally relevant concentrations of these herbicides. The developed method was also employed to analyze sample extracts collected during a passive sampling campaign in surface waters. The obtained data correlated well with results from LC-MS/MS chemical analysis but also revealed unknown PSII-inhibiting activities. The proposed methodology represents a rapid and sensitive screening tool for the simultaneous effect-based detection of PSII-inhibitors in environmental samples.