Serotonin is a key neurotransmitter involved in numerous physiological processes and serves as an important precursor for manufacturing bioactive indoleamines and alkaloids used in the treatment of human pathologies. In humans, serotonin sensing and signaling can occur by 12 G protein-coupled receptors (GPCRs) coupled to Gα proteins. In yeast, human serotonin GPCRs coupled to Gα proteins have previously been shown to function as whole-cell biosensors of serotonin. However, systematic characterization of serotonin biosensing modalities between variant serotonin GPCRs and application thereof for high-resolution serotonin quantification is still awaiting. To systematically assess GPCR signaling in response to serotonin, we characterized reporter gene expression at two different pHs of a 144-sized library encoding all 12 human serotonin GPCRs in combination with 12 different Gα proteins engineered in yeast. From this screen, we observed changes in the biosensor sensitivities of >4 orders of magnitude. Furthermore, adopting optimal biosensing designs and pH conditions enabled high-resolution high-performance liquid chromatography-validated sensing of serotonin produced in yeast. Lastly, we used the yeast platform to characterize 19 serotonin GPCR polymorphisms found in human populations. While major differences in signaling were observed among the individual polymorphisms when studied in yeast, a cross-comparison of selected variants in mammalian cells showed both similar and disparate results. Taken together, our study highlights serotonin biosensing modalities of relevance to both biotechnological and potential human health applications.