Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.