Quantitative single-particle analysis has revolutionized the study of biomolecules by enabling the detection and quantification of individual particles with high sensitivity and specificity. This chapter explores the application of surface-based fluorescence intensity distribution analysis (sFIDA) as a single-particle analysis method. Initially developed for the absolute quantification of protein aggregates, sFIDA integrates immunoassay principles with high-resolution fluorescence microscopy to achieve sub-femtomolar sensitivity. We highlight its use in diverse applications, including the analysis of misfolded protein oligomers in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Key features of the method, including selection of antibodies, high-throughput imaging of microtiter plates, and robust analytical workflows, are discussed. The potential of sFIDA as a diagnostic tool is exemplified through its ability to quantify aggregated amyloid-beta (Aβ), Tau, and α-synuclein in body fluids, providing critical insights into disease progression. Furthermore, we address its emerging applications in noninvasive matrices like plasma and feces, which hold promise for advancing biomarker discovery and therapeutic monitoring. This chapter provides a comprehensive guide to implementing sFIDA, from assay setup to data analysis, underscoring its potential to transform research and clinical diagnostics in precision medicine.