Crustaceans have been long used as model animals for neuromodulation studies because of their well-defined neural circuitry. The identification of small molecule metabolites and signaling molecules in circulating fluids and neuronal tissues presents unique challenges due to their diverse structures, biological functions, and wide range of concentrations. LC combined with high resolution MS/MS is one of the most powerful tools to uncover endogenous small molecules. Here we explored several sample preparation techniques (solid-phase extraction and denaturing) and MS data acquisition strategies (data-dependent acquisition and targeted MS2-based acquisition) that provided complementary coverage and improved overall identification rate in C18 LC-MS/MS experiment. By MS/MS spectral matching with mzCloud database and those generated from standard compounds, a total of 129 small molecule metabolites and neurotransmitters were identified from crustacean hemolymph and neuronal tissues. These confidently identified small molecules covered predominant biosynthetic pathways for major neurotransmitters, validating the effectiveness of the high-throughput RPLC-MS/MS approach in studying the metabolism of neurotransmitters.