Thousands of man-made chemicals are constantly released into the environment by agricultural and industrial production processes, traffic, and countless other human activities. Hence, very complex mixtures of anthropogenic chemicals and the transformation products of non-persistent compounds can be found in the aquatic environment. They reflect regional input but are also influenced by long-range transport. Thus, predicting effects on organisms and assessing the quality of a specific ecosystem based on chemical analysis is a challenge. This is not only because of the wide variety of chemicals, with far ranging physicochemical properties, but also because of the sometimes very low effect levels and concerted effects caused by concentration additivity or even synergism. The situation is further complicated by the temporal variability of the exposure concentrations caused, for example, by rain events or regular daily fluctuations as seen in wastewater treatment plant effluents. The analysis of an organism's proteome allows the detection of subtle changes in the level of individual proteins in response to environmental stressors. This potentially leads to the discovery of biomarkers of exposure and helps to gain insights into underlying mechanisms of toxicity. Today, studies using environmental proteomics have investigated many organisms, ranging from microorganisms and plants to invertebrates and vertebrates. Nevertheless, proteomics is a field of environmental research still in its infancy, due to a number of caveats, such as the limited number of organisms fully covered in the sequence databases, the high genetic variability, and the dependence on environmental factors. However, it is gradually becoming an established technology. This review article highlights recent advances in the field of proteomics, mainly focusing on experimental techniques that have the potential to help us understand toxic modes of action and identify novel ecotoxicological biomarkers.