Vibrio parahaemolyticus is a significant human pathogen associated with gastroenteritis. Two V. parahaemolyticus type 3 secretion systems, T3SS-1 and T3SS-2, secrete effector proteins and have been implicated in host-cell cytotoxicity and enterotoxicity, respectively. Vibrio parahaemolyticus T3SS-1 substrates have been identified, although many predicted substrates (based on homologies) remain undetected in secreted fractions and therefore uncharacterized. We have experimentally developed and optimized a secretion assay protocol allowing for reliable and reproducible detection of V. parahaemolyticus T3SS-1 secreted proteins within culture supernatants. The presence of magnesium and absence of calcium were critical factors in promoting type III secretion of protein substrates. Proteomic approaches identified known V. parahaemolyticus secreted effectors in addition to previously unidentified proteins. Isogenic mutants in putative low calcium response genes were generated, and experiments further implicated the genes in secretion and V. parahaemolyticus-mediated host-cell cytotoxicity during infection. These approaches should be valuable towards future detailed genetic and biochemical analyses of T3SS-1 in V. parahaemolyticus.