Advancements in proteomics and biomarker discovery require technologies capable of high-throughput, multiplexed, and minimally invasive protein detection. The Proximity Extension Assay (PEA), developed by Olink Proteomics, meets these demands by integrating dual-recognition immunoassays with quantitative PCR (qPCR), enabling the precise quantification of multiple proteins with exceptional sensitivity and specificity. While PEA is commonly applied to serum and plasma, its adaptability allows for the analysis of alternative biological matrices, including cerebrospinal fluid, urine, synovial fluid, bone marrow, amniotic fluid, blister fluid, conditioned media, interstitial fluid, and extracellular vesicles, among others. These matrices offer valuable opportunities for biomarker discovery, precision medicine, and translational research, particularly in diseases where conventional sampling is challenging. This chapter details the methodological considerations required for processing these alternative biofluids, ensuring their compatibility with PEA technology. It provides best practices for sample collection, processing, and quality control to maximize the accuracy and reproducibility of proteomic analyses. Additionally, it highlights PEA’s ability to detect low-abundance proteins, which is critical for identifying novel biomarkers in complex biological samples.