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Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca²⁺, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion <i>Hormurus waigiensis</i> to determine the breadth of Ca²⁺ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca²⁺ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca²⁺ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca²⁺ mobilization. <i>H. waigiensis</i> venom Ca²⁺ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.