An analytical method was developed to measure cis-permethrin and trans-permethrin in different biological rat matrices and fluids (whole blood, red blood cells, plasma, brain, liver, muscle, testes, kidneys, fat and faeces). The method was also suitable for the simultaneous quantification of their associated metabolites [cis-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis-DCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (trans-DCCA) and 3-phenoxybenzoic acid (3-PBA)] in blood (whole blood, red blood cells, plasma) and liver. The target analytes were derivatised in samples using a methanolic/hydrochloric acid solution and then extracted with toluene. The analysis was performed by gas chromatography, and detection using ion trap tandem mass spectrometry. The selectivity obtained for complex matrices such as rat organs allowed the use of a purification step to be avoided for most of the matrices investigated. In the case of fat, where permethrin is suspected to accumulate, a dedicated purification step was developed. In fluids, the limits of quantification were at the 50 ng/mL level for the parent compounds and 3-PBA and at 25 ng/mL for cis-DCCA and trans-DCCA. For solid matrices excluding fat, the limits of quantification ranged from 50 ng/g for muscle to 100 ng/g for brain and testes for both cis-permethrin and trans-permethrin. The extraction recoveries ranged primarily between 80 and 120% for the matrix tested. The stability of blood samples was tested through the addition of 1% v/v formic acid. The methods developed were applied in a toxicokinetic study in adult rats. cis-Permethrin and the metabolites were detected in all corresponding matrices, whereas trans-permethrin was detected only in blood, plasma and faeces.