Many bacteria utilize cell-to-cell communication systems that rely on small diffusible signal molecules to monitor the size of their population in a process known as quorum sensing (QS). QS plays a central role in coordinating genes that are generally mediating prokaryotic interactions with its eukaryotic host. In pathogens, this form of gene regulation is, for instance, believed to ensure that the cells remain invisible to the immune system until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish an infection. This review summarizes proteome analyses to identify QS-regulated proteins focussing on Gram-negative bacteria interacting with their eukaryotic hosts either as symbionts or as pathogens. In most studies, the power of comparative 2-D PAGE coupled to MS analysis has been employed to recognize and identify QS-controlled proteins. The high number of QS-regulated proteins in the majority of the investigated species strongly supports the importance of QS as global regulatory system and suggests that it also operates via post-transcriptional mechanisms. As QS has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in various opportunistic pathogens, it represents a highly attractive target for the development of novel antibacterial drugs. Proteomics has also been exploited to validate the target specificity of natural and synthetic QS inhibitors that have a great potential as alternative therapeutics for the treatment of bacterial infections.