Age-related macular degeneration (AMD) is a common form of adult blindness. Risk of developing AMD is strongly linked to genetics, in particular at the Regulators of Complement Activation (RCA) locus on Chr1, which contains 6 genes, Complement Factor H (CFH) and Complement Factor H-related 1-5 (CFHR1-5). These encode seven distinct proteins, since CFH gives rise to both a full length Factor H (FH) and a small splice variant, FHL-1 protein. These proteins act as fluid-phase co-factors for the breakdown of C3b and hence regulation of the complement cascade. Many of these SNPs are intronic; there is speculation that they modify the systemic levels of these proteins, causing an imbalance on complement regulation which impacts AMD development. Since FH, Factor H-related (FHR1-5) and FHL-1 proteins exhibit a high degree of homology, there is no antibody-based assay available for the simultaneous quantification of all seven related proteins. Here we describe a novel mass spectrometry-based assay for the simultaneous detection and quantification of all seven highly homologous human complement proteins; FH, FHR1, FHR2, FHR3, FHR4, FHR5 and FHL-1, in plasma. Since proteolysis using trypsin is not amenable to the generation of reliable proteotypic peptides for all seven proteins, we utilised Endoproteinase Glu-C (V8 Protease) as an alternative. We were able to generate unique signature peptides for all seven of the target proteins in a simple protocol which requires no pre-fractionation or clean-up stages during sample preparation. The method described is shown to be capable of the robust, simultaneous detection and relative quantification of all seven proteins with linearity and limits of detection appropriate to physiological levels. Once validated, the method was successfully applied to a clinical set of AMD control samples and compared to an existing FH antibody-based approach.