AmyloidosisAmyloidosis comprises a heterogeneous group of disorders marked by the extracellular deposition of insoluble protein fibrils, known as amyloids, which can disrupt normal tissue architecture and lead to organ dysfunction. Accurate identification and subtyping of the amyloidogenic protein are critical for clinical management as treatment strategies vary significantly depending on the underlying protein species. While conventional diagnostic tools such as Congo red staining and immunohistochemistry are commonly used, they suffer from limited specificity and antibody availability, often resulting in misclassification. Mass spectrometry (MS)-based proteomics has become the gold standard for amyloid subtyping, offering unmatched sensitivity and proteome-wide coverage. Here, we describe a spatial proteomics protocol that integrates laser capture microdissectionLaser capture microdissection (LCM) (LMD) with advanced LC-MS/MS acquisition methods, including data-dependent acquisition (DDA)Data-dependent acquisition (DDA), data-independent acquisition (DIA)Data-independent acquisition (DIA), and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMSField Asymmetric Ion Mobility Spectrometry (FAIMS)). With the integration of these methods, we present the analysis of formalin-fixed paraffin-embedded (FFPE)Formalin-fixed paraffin-embedded (FFPE) tissue sections. This comprehensive workflow enables precise excision of amyloid-rich regions and enhances detection of amyloid proteins and co-deposited biomarkers from minimal tissue input. The combination of FAIMSField Asymmetric Ion Mobility Spectrometry (FAIMS) with DIAData-independent acquisition (DIA) and DDAData-dependent acquisition (DDA) not only improves the depth of proteomic coverage but also increases reproducibility and sensitivity, making it particularly suitable for low-abundance samples. This protocol provides a robust and scalable platform for the accurate molecular subtyping of amyloidosisAmyloidosis and has the potential to inform personalized therapeutic decisions in clinical pathologyPathology.