Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT6 receptor (5- HT6R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT6R are poorly understood due to the lack of selective 5-HT6R ligands. In the present study, we examined functional coupling of the human 5-HT6R, 5-HT7AR, or 5-HT7BR with various Gα-proteins (Gα15, Gαqs5, or GαqG66Ds5) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT6R with GαqG66Ds5 produced the highest levels of Ca2+ signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT6R HTS system (Z´-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT6R, we screened ∼500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC50 value of 6.3 μM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT6R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT6R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT6R interacting protein. Taken together, these results suggest that the 5-HT6R/GαqG66Ds5 FDSS6000 system can be utilized to screen for selective 5-HT6R ligands and to examine any functional relationships between 5-HT6R and its binding proteins.