The structural novelty of lead compounds is very important in the agrochemical and pharmaceutical industries, and as such, natural products can be an important source. Taking into account that the isolation of lead compounds is very time-consuming, the efficient and safe identification of compounds in microorganism and plant extracts isolated previously is essential. A suitable procedure for this task based on an HPLC system interfaced with an electrospray (ESI) source and a Thermo Finnigan LCQ deca XP plus ion-trap mass spectrometer was developed, and an extensive MS/MS spectral library of characterized natural products was built up. This report summarizes the parameters used for acquiring the library spectra and discusses current limitations of the NIST library and search algorithm. The advantages of the newly introduced Mass Frontier 4.0 for the search of MS/MS product-ion spectra are discussed. Different mechanisms for fragmentation of some [M + H](+) and [M + Na](+) ions that were found are proposed. Oligomycin A, a macrolide antibiotic, exhibits different fragmentation mechanisms in positive and negative ion modes. The cleavage of the ester bond is the preferred mechanism in the positive ion mode, whereas two different pathways-one showing a rare retro-Michael-addition-are observed in the negative ion mode.