Active targeting nanoparticles are a new generation of drug and gene delivery systems with the potential for greatly improved therapeutics delivery compared to conventional nanomedicine approaches. Despite their potential, the translation of active targeting nanoparticles faces challenges in production scale-up and batch consistency. Accurate quality control methods for nanoparticle therapeutic payload and coating characterization are critical for attaining the desired levels of batch repeatability, drug/gene loading efficiency, targeting molecule coating effectiveness, and safety profiles. Current limitations in nanoparticle characterization technologies, such as relying on ensemble-average analysis, pose challenges in assessing the drug/gene content and surface modification heterogeneity, which can greatly affect therapeutic outcomes. Single-molecule analysis technologies have emerged as a promising alternative, offering rich information on heterogeneity and stochastic variations between nanoparticle batches. This review first evaluates and identifies the challenges of traditional nanoparticle characterization tools that rely on indirect, bulk solution quantification methods. Subsequently, newly emerging characterization technologies are introduced for the quantification of therapeutic loading and targeted moiety coating efficiencies with single-nanoparticle resolution, to help guide researchers towards advancing the translation of active targeting nanoparticles into the clinical setting.