Nocturnal outdoor illumination is mainly designed aiming at human needs. However, side effects on other organisms and ecosystems have to be expected, for example for fish populations. Basic knowledge about biological action of light on fish could be gained from laboratory experiments. This work presents an experimental strategy to study the biological action of light on fish. It is found that the melatonin expression of European perch (Perca fluviatilis) is significantly suppressed at an illuminance level of 100 lx throughout the night. The experimental approach is described in detail including biological methods, experimental illumination design and mathematical modeling for reconstruction of melatonin release rates from water melatonin concentrations.