Mycotoxins are harmful food contaminants. Currently, human exposure assessment to these toxins is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, biomarkers have been proposed as a suitable alternative whereby a more accurate assessment of exposure at the individual level can be performed. The BIOMYCO study is designed to assess human mycotoxin exposure using urinary biomarkers of exposure. Over the different seasons of 2013 and 2014, morning urine is gathered in a representative part of the Belgian population according to a designed study protocol, whereby 140 children (3–12 years old) and 278 adults (19–65 years old) are selected based on random cluster sampling stratified for sex, age and geographical areas. Every participant completes a food frequency questionnaire to assess the consumption of relevant foodstuffs (n = 43) of both the day before the urine collection and the previous month. Validated multi-toxin LC-MS/MS methods are used to analyse aflatoxins, fumonisins, ochratoxin A, trichothecenes, zearalenone and their metabolites in morning urine. The study protocol is approved by the ethical committee of the Ghent University Hospital. Within this paper, study design and methods are described. The BIOMYCO study is the first study whereby a multi-toxin approach is applied for mycotoxin exposure assessment in adults and children on a large scale. Moreover, it is the first study that will describe the exposure to an elaborated set of mycotoxins in the Belgian population. In first instance, descriptive analysis will be performed, describing the exposure to mycotoxins for the child and adult group. Exposure of different subgroups will be compared. Furthermore, correlations between the mycotoxin concentrations measured and the food consumption reported will be estimated to explore whether the mycotoxin exposure could be explained by the consumption of certain foods.