The synthetic pyrethroid permethrin has widespread use in agriculture and, as a result, is found in a variety of matrices. Chemical methods based on gas chromatography/mass spectrometry operated in the negative chemical ionization mode (GC‐NCI/MS) have been developed. Sample extraction techniques such as ultrasonication and steam distillation have also been examined. Significant improvements have been made, compared with traditional methods, in both sample extraction time and detection level for a range of matrices. Enzyme‐linked immunosorbent assay (ELISA) methods have also been developed for permethrin. This study addresses the comparison of ELISA and chemical assays for permethrin in a variety of both environmental and laboratory‐spiked samples. The sensitivities, specificities and potential applications of these methods are discussed.