Mycotoxins are toxic secondary fungal metabolites that frequently contaminate cereal crops globally, presenting exposure hazards to humans and livestock in many settings. The heterogeneous distribution of mycotoxins in food restricts the usefulness of food sampling and intake estimates for epidemiological studies, making validated exposure biomarkers better tools for informing epidemiological investigations. While biomarkers of exposure have served important roles for understanding the public health impact of mycotoxins such as aflatoxins (AF), the science of biomarkers must continue advancing to allow for better understanding of mycotoxins’ roles in the etiology of disease and the effectiveness of mitigation strategies. This review will discuss mycotoxin biomarker development approaches over several decades for four toxins of significant public health concerns, AFs, fumonisins (FB), deoxynivalenol (DON), and ochratoxin A (OTA). This review will also highlight some knowledge gaps, key needs and potential pitfalls in mycotoxin biomarker interpretation.