Human exposure to mycotoxins is almost inevitable as mycotoxins are naturally occurring contaminants of large portion of food and feed. Depending on the type of mycotoxins, inter-individual mycotoxin adsorption, bioaccumulation, distribution, metabolism and excretion, can cause serious adverse health effects. Therefore, continuous biomonitoring studies of population exposure to mycotoxins are needed. Here we describe a multi-analyte approach for the detection and quantification of 20 mycotoxins in human serum using ultra-performance liquid chromatography-electrospray/tandem mass spectrometry operated in targeted multiple reaction monitoring mode. The validated method was used to assess occurrence of mycotoxins in serum samples of 46 residents of Qatar. Mycotoxins that were detected with high incidence were HT-2 toxin (13.0%), sterigmatocystin (10.9%) and 3-acetyldeoxynivalenol (6.5%). Also, co-exposure to several mycotoxins was noticed in the analysed samples. Our results show that strict food quality control is needed to remove mycotoxin contaminated food from the market in order to minimise human exposure to mycotoxins.