The 5-HT1A and 5-HT1B serotonin receptors are abundantly expressed in the CNS and constitute validated as well as putative drug targets in a variety of psychiatric and cognitive disorders, alcoholism/addiction, pain and migraine. In the present study we have characterized the functional properties of human 5-HT1A and 5-HT1B stably co-expressed with the human G-protein-activated inwardly rectifying K+ channel 2 (GIRK2) in HEK293 cells in the fluorescence-based FLIPR® Membrane Potential Blue (FMP) assay. Serotonin and other agonists induced robust decreases in fluorescence levels in the 5-HT1A/GIRK2- and 5-HT1B/GIRK2-HEK293 cells in a concentration-dependent manner in the assay, and these responses could be inhibited by selective 5-HT1A/5-HT1B antagonists and by the Gαi/o-protein inhibitor pertussis toxin (PTX). Five additional stable HEK293 cell lines co-expressing 5-HT1A or 5-HT1B with GIRK2 and one of the PTX-insensitive Gαi/o-subunit mutants Gαi1C351I, Gαi2C352I and Gαo1C351I were constructed, and 5-HT1A/5-HT1B-mediated responses through these specific Gαi/o-subunits were measured in these cells pretreated with PTX in the FMP assay. The functional properties of 16 reference 5-HT1 agonists were characterized at the seven cell lines, which constitutes the most detailed pharmacological profiling and comparison of 5-HT1A and 5-HT1B receptor signaling in the same assay published to date. We propose that this approach to assay 5-HT1-mediated signaling through endogenous Gαi/o-proteins in HEK293 cells or through specific Gαi/o-subunits in a fairly high-throughput manner holds some advantages to other functional assays for Gαi/o-coupled receptors. The assay will facilitate detailed profiling of the Gαi/o- and Gβγ-mediated signaling of 5-HT1A and 5-HT1B at the molecular level, and it could also be used to identify novel modulators for the receptors. Copyright © 2020 Elsevier Inc. All rights reserved.