The river analyzer (RIANA) is an optical immunosensor able to determine different types of organic pollutants in water samples. Interaction between fluorescently labeled antibody and analyte takes place on a solid-liquid interface and is detected by measuring the fluorescence. The RIANA integrates the optical part with a fluidics part in a compact unit, which is coupled to an autosampler, allowing the performance of automatic measurements within 15 min. In this chapter, the use protocol for the sensor is described, and guidelines about experiment performance are provided. Up to three different analytes (in this case, the pesticides atrazine and isoproturon and the natural estrogen estrone) can be detected simultaneously. This multianalyte configuration allows reduction in time of analysis and in volume of sample and other reagents required. Different aspects, such as assay performance, cross-reactivity, matrix effects, or validation, are also considered.