Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of microorganisms has shown its usefulness in clinical laboratories, but there are some important limitations associated with this technique. In this study, we present proof of concept of a newly developed assay which can resolve most of these MALDI-TOF MS limitations. This assay is based upon multiplex liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the targeted identification of different species in mixed samples. Four clinically relevant species of the fungal genus Aspergillus were used in this proof-of-concept study, and mixed samples were artificially prepared with strains of verified identity. The principle of the LC-ESI-MS/MS assay was based upon the detection of targeted Aspergillus species/genus-specific proteotypic peptide markers. Both pure material (i.e., single strains) and artificially mixed Aspergillus samples (with up to four constituents) were tested. Thirty selected Aspergillus species/genus-specific proteotypic peptides were applied in our LC-ESI-MS/MS assay after optimization. An overall success rate of 85 and 92.3% correct identification was obtained when testing the pure material and the artificially mixed Aspergillus samples, respectively. In contrast to MALDI-TOF MS, only one of the constituents of the artificially mixed Aspergillus sample could be correctly identified, whereas a nearly 100% success rate was obtained when testing the pure material. In conclusion, we have developed an innovative LC-ESI-MS/MS assay which has the capacity to determine the composition of mixed samples, and its proof of concept is shown here for Aspergillus species. This LC-ESI-MS/MS assay breaks new ground as a rapid and reliable tool for the identification of fungi and other microorganisms in clinical laboratories, even when dealing with samples in which the presence of multiple species is suspected.