To date, 11 gene loci that contribute to familial Parkinson's disease (PD) are known. Of these, mutations in six genes have been identified, allowing genetic testing and more accurate phenotypic characterization of genetically defined disease subtypes. In particular, mutations in Parkin, DJ-1, and Pink1 genes are associated with autosomal recessive PD and may also play a major role in early onset PD (EOPD). However, genetic testing for sequence alterations in these genes remains laborious. Therefore, our aim was to develop a flexible, rapid, high-throughput screening procedure using matrix-assisted laser desorption ionization/time of flight technology and homogeneous mass cleave assays. Using this novel approach, we screened all 27 coding exons of the Parkin, DJ-1, and Pink1 genes in 31 patients with EOPD, a total of 367,195 nucleotides. Four positive controls with known autosomal recessive PD mutations that had previously been screened by denaturing high performance liquid chromatography in combination with sequencing were also tested. All known alterations were detected by matrix-assisted laser desorption ionization/time of flight mass spectrometer, as well as additional polymorphisms in formerly unscreened regions. Overall, two previously described mutations in three patients with EOPD, 27 known polymorphisms with 386 occurrences, and eight unknown variants with 21 occurrences were detected. In total, we identified 410 sequence alterations in 31 patients with EOPD. In conclusion, this is the first study using matrix-assisted laser desorption ionization/time of flight mass spectrometry and homogeneous mass cleave assay for high-throughput mutation screening.