A bioanalytical assay for the new tyrosine kinase inhibitor axitinib was developed and validated. In addition, the light mediated trans to cis isomerization of this drug was investigated. For the quantitative assay, human plasma samples were pre-treated under light protection using protein precipitation with acetonitrile containing erlotinib as the internal standard. The extract was diluted with water and injected into the chromatographic system. The system consisted of a trifunctional bonded octadecyl silica column with isocratic elution using formic acid in a water-methanol mixture. The eluate was transferred into an electrospray interface with positive ionization and the analyte was detected and quantified using the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.2-200ng/ml concentration range, the lowest level of this range being the lower limit of quantification. Within day precisions were 2.5-6%, between day precisions 4-9% and accuracies were between 91 and 106% for the whole calibration range. Light protected axitinib showed no isomerization and was shown to be chemically stable under all relevant conditions. Finally, the assay was successfully applied for a mouse tissue distribution study using mouse samples diluted with human plasma.