Infections with organisms that are difficult to culture have been historically diagnosed by serological methods. More recently, molecular methods have become available to the clinical immunology and microbiology laboratories. These methods include variations on DNA and RNA amplification techniques such as the polymerase chain reaction. These methods offer increased sensitivity and become positive earlier in an infection than antibody tests. Unfortunately, the molecular methods are costly and offer the possibility of false-positive results. This article provides a review of serological and molecular methods available for diagnosing and monitoring human immunodeficiency virus (HIV), Borrelia burgdorferi, and West Nile virus infections. With the exception of neonatal HIV diagnosis, antibody detection methods are used for routine diagnosis. Molecular methods have proven useful, especially in the case of HIV, for monitoring response to treatment.