Familial hypercholesterolaemia (FH) is a common single gene disorder, pre-disposing to cardiovascular disease, which is most commonly caused by mutations in the LDL-receptor (LDLR) gene. About 5% of patients carry the p.R3527Q (previously R3500Q) mutation in the apolipoprotein B (APOB) gene and 2% carry the p.D374Y mutation in the PCSK9 gene, but the lack of high-throughput methods make routine genetic diagnosis difficult. In this study, we developed an iPLEX MassARRAY Spectrometry mutation test to identify 56 mutations (54 in the LDLR gene, 1 in the APOB gene and 1 in the PCSK9 gene). The iPLEX test was verified by analysing 150 DNA samples from FH patients with a previously characterized mutation and 96 no-mutation control samples. Mutations were identified in all 150 FH mutation-positive samples using the iPLEX assay, with 96% directly called by the software. The false-positive rate in no-mutation control samples was 0.015%. The overall specific mutation assay failure rate was 2.1%. In the UK, this gives an average detection rate of 75%.The FH iPLEX test is not only designed for large-scale targeted population screening for FH mutations, such as lipid clinic patients, but can also be used for population screening. The assay can easily be developed further to include additional FH-causing mutations, thus increasing the sensitivity of the diagnostic assay.