The gut-brain-axis (GBA) is a bi-directional communication system between the gastrointestinal (GI) enteric nervous system and the central nervous system, capable of complex crosstalk between the gut and the brain to maintain GI homeostasis and influence mood and higher cognitive functions. Under healthy conditions, this communication is beneficial for regulating immune function, proper peristaltic motion, and hormone release related to hunger and feeding behaviors. However, GBA communication can cause co-morbid occurrence of both GI and neural disorders. For instance, chronic inflammatory conditions of the gut, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), often present with symptoms of depression and anxiety. Clinical studies, animal models, and molecular research techniques have implicated serotonin (5-HT) as a key signaling molecule to both regulate GI functions and stimulate enteric nerves. These studies are limited by the inability to study sub-mucosal 5-HT on the basolateral side of the epithelium, wheremost of the 5-HT is released and acts on nerves endings. The ability to measure 5-HT release patterns in this area, at native spatial and temporal scales, within an in vitro culture of the gut epithelium, would allow researchers to distinguish 5-HT release patterns stimulated by different GI luminal conditions associated with health and disease, to better understand how these stimuli affect the brain. In this dissertation, electrochemical sensors are fabricated within two types of in vitro platforms to measure 5-HT at physiological scales (sub-micromolar concentrations). The goal of this design is to facilitate the direct detection of 5-HT released from cells cultured in the platform to improve both spatial and temporal access to basolaterally-secreted molecules and provide continuous, automated measurements over experimental time scales. 5-HT sensors fabricated on both porous and smooth cell culture substrates are demonstrated, achieving sensitivities of ~1 – 10 μA/μM and limits of detection of ~100 nM. Electrochemical characterization allow understanding of 5-HT adsorption kinetics, which was modeled to track and predict sensor fouling over continuous measurements. These sensor-integrated substrates were packaged in 3D printed structures, which allowed rapid fabrication of custom designs and were shown to be biocompatible and support growth of RIN14B cells, a model 5-HT-secreting cell line. Finally, cell-secreted 5-HT was detected at ~100 – 500 nM, corresponding to ~4 pmol 5-HT / 105 cells. Ultimately, slow adsorption kinetics prevented direct detection of 5-HT from cells cultured directly on top of the sensors, but the thorough characterization of the platform demonstrated here lays significant groundwork for future optimization of the sensing protocol.