Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFP-guided single-cell microdissection, on-plate chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) or 2,5-dimethyl-1 H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDI-TOF MS. We were able to determine a limit of detection for OA of 1 fmol/μL, for TA of 2.5 fmol/μL and a lower limit of quantification (LLOQ) of 10 fmol/μL for both substances. SCMS of GFP-labeled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/μL and a TA titer (∼2.5 fmol/μL) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2 > GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.