Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof;​ additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.