Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here, we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well-known nephrotoxicant. Differential analysis of the mass spectrum profiles reveals a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (S₂₈-Q₁₄₆) using an innovative combination of tissue micro-extraction, fractionation by reverse-phase liquid chromatography, followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of IMS in the identification of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.