Proper function of the cardiac ryanodine receptor type 2 (RyR2) is crucial for normal rhythmicity and performance, especially during adrenergic activation. RyR2 gating is under stringent control of the calcium induced calcium release mechanism and restricts global increases of [Ca²⁺]_i to the systolic phase. Numerous human RyR2 mutations are related to catecholaminergic polymorphic ventricular tachycardia (CPVT), characterized by arrhythmogenic attacks, often with sudden cardiac death, under physical or emotional stress. We recently characterized the novel RyR2 mutation F2483I in patient-derived, cardiac-type adult stem cells (iPSC), revealing irregularities in excitability and Ca²⁺ handling. However, iPSC can be functionally heterogeneous, which sometimes precludes a straightforward relation of experimental observations to the mutation. Here, we describe a method for functional screening of disease-related RyR2 variants in a heterologous system (HEK293), allowing simultaneous comparison of Ca²⁺ release kinetics/sensitivities between wild type (wt) and mutant channels, in the same assay. We generated plasmids for the expression of RyR2^wt and RyR2^F2483I, their N-terminal labelling with either cyan or red fluorescent protein facilitates spectral separation between the RyR2 species and from the signal of the green fluorescent Ca²⁺ indicator Fluo-4. After transfection with different plasmids in separate cultures, the cells are detached and plated on the same dish. Upon application of RyR activators/modulators (e.g. caffeine, 4-CMC, [Ca²⁺], ryanodine, 8-Br-cAMP), Ca²⁺ release in cells expressing either RyR2^wt and RyR2^F2483I is measured within the same microscopic filed of view. This new approach reduces the influence of varying experimental conditions and allows us to reveal even subtle mutational changes in RyR2 function.