Protein-conjugated gold nanoparticles (AuNPs) have been extensively explored for the development of many novel protein assays. In this article, we demonstrate that nanoparticle tracking analysis (NTA) can be used as a rapid and simple analytical tool to monitor bioconjugation and to study protein–protein interactions. The adsorption of protein A onto gold nanoparticles was analyzed using NTA. The conjugation resulted in a measurable increase in hydrodynamic radius that correlated with protein A concentration, allowing conditions for complete conjugation to be elucidated. NTA was then used to investigate the binding of mouse IgG to protein A-conjugated AuNPs and the K_a was measured as 2.00 × 10⁷ M⁻¹. Furthermore, an assay for the detection of mouse IgG was developed using NTA to detect the binding to antibody–AuNP conjugates. This assay provided a detection limit of 3.2 ng mL⁻¹; however, the formation of aggregates resulting from the use of a polyclonal antibody and multiple binding sites on the antigen prevented the determination of binding affinity for this antibody–antigen system. To measure the binding affinity for this antibody–antigen system the IgG antigen was conjugated to the AuNPs and NTA was used to monitor the binding of the antibody. In this configuration aggregation of conjugates was not detected and a binding affinity constant of 2.80 × 10⁸ M⁻¹ was measured. NTA results obtained in this work were validated by comparison to DLS. This work represents the first evaluation of NTA as an analytical tool for characterizing AuNP bioconjugates, investigating protein–protein binding, and detecting low levels of antigen in a bioassay.