EAI045 is a tyrosine kinase inhibitor (TKI) that targets the mutant epidermal growth factor receptor (EGFR). It was developed to control resistance to available EGFR TKIs. In this study, a major metabolite of EAI045, (5-fluoro-2-hydroxyphenyl)(1-oxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid (PIA), was discovered as a hydrolysis product of the parent drug. A validated assay for both analytes in mouse plasma and tissue homogenates from brain, kidney, liver, lung, spleen, and small intestine with content was set up using LC-MS/MS. Samples were prepared by protein precipitation with acetonitrile and with PLX4720 as internal standard. Separation was performed on a bridged ethylene hybrid C18 column by gradient elution with 0.1% v/v formic acid and methanol. Using positive electrospray, detection was performed in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml was used and validated for both analytes. Precision values ranged between 2.0 and 7.5% for EAI045 and between 2.2 and 12.1% for the metabolite, and accuracy values were between 91.1 and 107.6% for EAI045 and between 87.6 and 100.6% for the metabolite. Both analytes were sufficiently stable under the relevant analytical conditions. Finally, the assay was applied to analyze mouse plasma and tissue levels in a pharmacokinetic study in FVB/NRj wild-type female mice treated with oral EAI045.