Formalin-fixed and paraffin-embedded (FFPEPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPEPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALALDI-FT-ICRCR-MSI) platform. The method involves FFPEPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALALDI-MSI. Using this platform, we previously detected similar to 1,500 m/z species in the mass range m/z 50-1,000 in FFPEPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPEPE tissue samples. This protocol can be reproducibly performed on FFPEPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.