For the first time a multiplexed PCR approach suitable for mass spectrometric STR allele identification is presented. Thirteen forensically important STR markers (vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, TPOX, CSF1PO, D2S441, D10S1248, and D22S1045) and the gender typing locus amelogenin were simultaneously amplified. Ion-pair reversed-phase high-performance liquid chromatography electrospray-ionization time-of-flight mass spectrometry (ICEMS) was applied for genotyping, and allowed for highly efficient characterization of multiple PCR amplicons. Compared with electrophoretic sizing ICEMS enabled for the simultaneous detection of length and nucleotide variations. Thus, the obtained amount of biological information present within STR profiles was significantly increased even though the compatibility of typing results with electrophoretically generated data(bases) was maintained. Other advantages of the ICEMS platform included the abandonment of internal size standards, allelic ladders, and any kind of spectral calibration. The 14-plex PCR was tailor-made for ICEMS analysis by designing primer pairs that bind close to the repeat region, by using a proof reading polymerase for amplification, and by implementing molecular mass modifiers for prevention of molecular mass overlaps. In a series of experiments, the performance of the multiplexed PCR-ICEMS assay was evaluated. The ICEMS-based DNA profiling assay was found to be competitive regarding detection sensitivity and analyzability of degraded and casework samples with commercially available electrophoretic typing approaches, which suggests that multiplexed PCR-ICEMS assays could represent a valuable tool for (forensic) genetics.