The ryanodine receptor Ca2+-release channels (RyRs) are central to cytoplasmic Ca2+ signalling in particular in the skeletal muscle of both mammals and insects. Regarding the latter, several insecticides target RyRs but recent studies have shown that specific RyR point mutations can confer insecticide resistance. Consequently, research for new RyR modulators remains crucial for crop protection. The full-length coding sequence of the Plutella xylostella RyR was synthesized and cloned into suitable mammalian expression vector. We engineered HEK-293 expressing the insect target and functionally selected stable clones by the use of Ca2+-sensitive fluorescence. In order to exclude any endogenous non-specific response, a related mock clone was also generated in parallel. Stimulation of the cells to activate the RyR elicited a Ca2+ mobilization, which was detected as a fluorescence increase using a FLIPR-TETRA instrument. This RyR cell-based assay was validated with RyR ligands and putative blockers and further optimized to make it suitable for high throughput screening. Drug discovery programs, which may involve the screening of >105 compounds, requires a high fidelity method of determining the effect of putative hits on the function of the target protein. Since RyR expression is targeted to internal membranes, patch-clamp of the nuclear membrane has been previously shown to be an effective method of isolating them for single-channel recording. We therefore set up this very challenging technique to measure the RyR activity single-channel properties (conductance, open probability -Po- and dwell time distribution) and to show the modulatory effect of Ca2+ on these above-mentioned parameters. From cell cloning to Mode of Action determination, we develop an assay funnel, optimized for hit sensitivity and specificity that is easily scalable to larger compound libraries.