Neuropeptides are an important class of signaling molecules in the nervous and neuroendocrine system, but they are challenging to study due to their low concentration in vivo in the presence of numerous interfering artifacts. Often the limitation of mass spectrometry analyses of neuropeptides in complex tissue extracts is not due to neuropeptides being below the detection limit but due to ions not being selected for tandem mass spectrometry during the liquid chromatography elution time and therefore not being identified. In this study, a data independent acquisition (DIA) method was developed to improve the coverage of neuropeptides in neural tissue from the model organism C. borealis. The optimal mass-to-charge ratio range and isolation window were determined and subsequently used to detect more neuropeptides in extracts from the brain and pericardial organs than the conventional data dependent acquisition method. The DIA method led to the detection of almost twice as many neuropeptides in the brain and approximately 1.5-fold more neuropeptides in the pericardial organs. The technical and biological reproducibility were also explored and found to be improved over the original method, with 56% of neuropeptides detected in 3 out of 3 replicate injections and 62% in 3 out of 3 biological replicates. Furthermore, 68 putative novel neuropeptides were detected and identified with de novo sequencing. The quantitative accuracy of the method was also explored. The developed method is anticipated to be useful for gaining a deeper profiling of neuropeptides, especially those in low abundance, in a variety of sample types.