Applying a proteomic approach for biomonitoring marine environments offers a useful tool for identifying organisms' stress responses, with benthic filter-feeders being ideal candidates for this practice. Here, we investigated the proteomic profile of two solitary ascidians (Chordata, Ascidiacea): Microcosmus exasperatus, collected from five sites along the Mediterranean coast of Israel; and Polycarpa mytiligera collected from four sites along the Red Sea coast. 193 and 13 proteins in M. exasperatus and P. mytiligera, respectively, demonstrated a significant differential expression. Significant differences were found between the proteomes from the northern and the southern sites along both the Mediterranean and the Red Sea coasts. Some of the significant proteins had previously been shown to be affected by environmental stressors, and thus have the potential to be further developed as biomarkers. Obtaining a proteomic profile of field-collected ascidians provides a useful tool for the early-detection of a stress response in ascidians worldwide.