Mycotoxins are toxic secondary metabolites produced by fungi, and are capable of causing disease and death in both humans and animals. Sample preparation is a critical step in the successful analysis of mycotoxins in food matrices. The “dilute and shoot” approach is a quick and easy way to introduce the sample into the LC/MS/MS analysis. However, due to the complexity of food matrices, this approach will often result in irreproducible matrix effects. To overcome these problems and improve sensitivity, this application note details the use of online solid phase extraction (SPE), coupled to an LC/MS/MS system for sample enrichment.