The Serotonergic System is a major component in the function and integration of brain circuitry. Serotonergic neurons have long cable-like structures called axons. The developmental process of axon elongation and branching is fundamental to the function of the nervous system (Gaspar et al., 2003). Studying the morphology, structure, and function of axons has been difficult due to limitation in traditional staining and imaging technologies. Expansion Microscopy and Brainbow imaging pose a new avenue to study these detailed axon morphologies. The protocol implemented through this experiment was developed to better enhance the advantages of these innovative technologies. This protocol is a non-genetic manipulation to increase serotonin, 5HT, in the Central Nervous System (CNS) of Drosophila melanogaster (Drosophila). Genetic approaches to manipulating serotonin exists, but do not allow either concentration or temporal variances to be studied. This goal of this experiment was to show that administering a 5HTPinfused food to the Drosophila larvae would increase the levels of serotonin in the 3rd instar Drosophila larvae Central Nervous System. In this protocol, a serotonin precursor, 5- hydroxytryptophan (5HTP), was added to the food during the 2nd instar larval stage to increase 5HT levels in the 3rd instar Drosophila larvae CNS. Through both immunohistochemistry and imaging analysis of pixel intensities, quantification of 5HT in the CNS of Drosophila was obtained for experimental (5HTP-infused food) and control groups. The results indicated this protocol increased the 5HT levels in the CNS of 3rd instar Drosophila larvae of the experimental groups with statistical significance. These results show that this protocol was successful and could possibly be used for further experimentation in Brainbow Drosophila. This allows for detailed study of changes in brain circuitry at varying levels of serotonin.