Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.