SIMOA is a highly sensitive immunoassay technology used for the ultrasensitive detection and quantification of specific biomolecules, typically proteins, in various biological samples. It utilizes antibody-coated beads and fluorescently conjugated detection antibodies that are applied within the samples to a specialized cartridge with microwells, allowing for single-molecule detection. Due to its ability to detect and quantify biomolecules at extremely low concentrations, SIMOA is used in medical and biological research, and diagnostics for various biomarkers, including those related to cancer, neurodegenerative diseases, and cardiovascular conditions. In this chapter, we described the method to monitor cerebrospinal fluid (CSF) biomarkers as a valuable tool in early disease detection and monitoring.