In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]+ ions.