The entomopathogenic fungus Metarhizium brunneum is widely applied as a biological pest control agent. Consequently, its use has to be accompanied by a risk management approach, which includes the need to monitor the fate of its bioactive metabolites in the environment, for example, in treated crops. A fast and selective UHPLC-QTOF-MS method was developed to monitor the presence of secreted destruxins in two model food plants for the application of this fungal biocontrol agent, namely, strawberry and maize. The liquid chromatography-mass spectrometric assay for destruxin trace analysis is combined with a novel QuEChERS-based extraction protocol. The whole assay was optimized for the application in these crops, and it allows quantitative analysis of the major M. brunneum metabolites destruxin A, 1, destruxin B, 2, and destruxin E, 3, down to the parts per billion range. In strawberry, limits of quantitation (LOQs) were found to be <2.0 ppb for all analytes; in maize LOQs were found to be <3.2 ppb for destruxin A and destruxin B. Destruxin E showed a distinctive loss of recovery in maize and was excluded from further quantitative analysis in this crop. For both crops assay linearities ranged from the LOQs to 100 ppb, interassay repeatabilities (RSD) were found to be better than 16.4%, and accuracies ranged from 83.5 to 105.3% (assessed at four spiking levels between 5 and 75 ppb).