Molecular diagnostics have the potential to revolutionize our ability to diagnose, monitor and treat a variety of diseases. However, detecting circulating biomarkers has proven to be challenging due to the low concentrations of many biomarkers. Furthermore, other factors including sample heterogeneity, low sample volume, and cross-reactivity between different markers complicate biomarkers detection. Consequentially, there is a need for improved biomarker detection methods. The emergence of single-molecule techniques opens new opportunities to overcome these challenges. Counting individual molecules enables the detection of biomarkers at low quantities. Additionally, single-molecule technologies are particularly relevant to the analysis of heterogeneous samples, and can allow for biomarkers detection with a wide dynamic range. The Walt lab previously developed an ultra-sensitive assay known as Single Molecule Arrays (Simoa) by isolating single molecules into femtoliter-sized compartments using microfluidics, and detecting them optically. The Simoa technology enables biomarkers to be measured at concentrations a thousand times lower than current molecular diagnostic methods, providing a more accurate approach for diagnosing certain forms of cancer, infectious diseases and neurodegenerative disorders. Recently, the COVID-19 pandemic has led to an urgent need for robust diagnostic assays. To address this need, we developed ultra-sensitive Simoa based methods for the detection of anti-SARS-CoV-2 antibodies and viral antigens, and an assay for the quantification of antibody neutralization capacity. We used these new assays to study important clinical problems in collaboration with clinicians in Brigham and Women’s Hospital and Massachusetts General Hospital. For example, we studied the mechanism of Multisystem Inflammatory Syndrome in Children (MIS-C). Our new findings on MIS-C pathogenesis provide novel targets for treating and preventing MIS-C, which are urgently needed for this increasingly common, severe COVID-19-related disease in children. The past year has expedited the use of Simoa in the clinic, and we are now excited about further developing and applying single molecule detection methods for numerous clinical applications.