Immunoassays, or assays that are using antibodies as the specific binding reagents, have become one of the most common methodologies in diagnostic laboratories. In this paper we review different configurations of immunoassays as applied to a variety of analytes and sensitivity limits, along with common detection techniques and strategies. Progress in developing of ultra high affinity antibodies as a direction to improved immunoassays is also reviewed. Finally, we specifically concentrate on determination of antibody binding constants and performing immunoassays at the single molecule level using Fluorescence Correlation Spectroscopy (FCS). This technique has become a powerful tool in molecular binding characterizations and assay development, and possibly will grow into a quantitative analytical method suitable for diagnostic tests.