The biosynthesis of neuropeptides involves multiple trimming steps and post translational modification, thus impeding the prediction of the active peptide from the genome sequence or even the precursor protein. Therefore we believe that the analysis of undigested neuropeptides by a reproducible and robust method such as SRM is vital to accurately quantify the biologically active compounds. In a proof of principle study we quantified 15 neuropeptides in a rat obesity model using Skyline to perform assay development and optimization. We could show, that despite common practice precursor and fragment charges of up to 5 and 3 respectively do not limit the quality of the SRM peak groups, however require a dedicated individual optimization of the collision energies.