There is a constant need to improve the performance of current diagnostic assays as well as develop innovative testing strategies to meet new testing challenges. The use of nanoparticles promises to help promote in vitro diagnostics to the next level of performance. Quantum dots (QDs), gold nanoparticles (AuNPs), and superparamagnetic nanoparticles are the most promising nanostructures for in vitro diagnostic applications. These nanoparticles can be conjugated to recognition moieties such as antibodies or oligonucleotides for detection of target biomolecules. Nanoparticles have been utilized in immunoassays, immunohistochemistry, DNA diagnostics, bioseparation of specific cell populations, and cellular imaging. Nanoparticle-based diagnostics may open new frontiers for detection of tumours, infectious diseases, bio-terrorism agents, and neurological diseases, to name a few. More work is necessary to fully optimize use of nanoparticles for clinical diagnosis and to resolve some concerns regarding potential health and environmental risks related to their use. However, we envision further developments of nanoparticle-based diagnostics will yield unique assays with enhanced sensitivity and multiplexing capability for the modern clinical laboratory.