The development and validation of a bioanalytical assay is described for the simultaneous analysis of 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) and its main metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in plasma, urine, faeces, bile, liver, kidney, testis, spleen, brain, as well as colon-, cecum- and small intestinal tissue and contents from mice. The effect of the matrix on the accuracy of the method was extensively investigated. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for analyte quantification. The assay is validated from 1 to 250 ng/mL and the sample pretreatment consists of protein precipitation with acetonitrile using only 100 microL matrix (plasma, bile diluted in 4% (m/v) BSA, intestinal contents, faeces and tissue samples homogenized in 4% (m/v) BSA). The measured concentrations of PhIP and N-OH-PhIP in homogenates were expressed in ng/mL. Based on the weight of the isolated intestinal contents, faeces or tissue the amount of PhIP and N-OH-PhIP per mass unit intestinal content, faeces or tissue was calculated. The validated range for PhIP in urine is from 10 to 1000 ng/mL using 20 microL urine. For N-OH-PhIP quantification, mouse urine was diluted 100 x in blank human urine to compensate for matrix effects. The developed method is simple, robust and reproducible. The applicability of the method was demonstrated and the assay could be successfully used to support in vivo toxicokinetics studies of PhIP and N-OH-PhIP in mice.