In-depth proteome exploration of complex body fluids is a challenging task that requires optimal sample preparation and analysis in order to reach novel and meaningful insights. Analysis of follicular fluids is similarly difficult as that of blood serum due to the ubiquitous presence of several highly abundant proteins and a wide range of protein concentrations. Therefore, the accessibility of this complex body fluid for liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis is a challenging opportunity to gain insights into the physiological status or to identify new diagnostic and prognostic markers for e.g. the treatment of infertility. We compared different sample preparation methods (FASP, eFASP and in-solution digestion) and three different data analysis software packages (Proteome Discoverer with SEQUEST and Mascot, Maxquant with Andromeda) in conjunction with semi- and full-tryptic databank search approaches in order to obtain a maximum coverage of the proteome.