Technical Abstract: An important mechanism of natural disease suppression is the production of antibiotics by soil microbes. Antibiotics are small organic molecules of microbial origin that at low concentrations are deleterious to the growth or metabolism of other microorganisms. Soilborne pathogens are sensitive to many antibiotics produced by soil microbes on the roots of plants. Phenazines, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, and pyoluteorin are some of the most common of these antibiotics, but they are just a small fraction of the thousands that comprise the rhizosphere metabolome. Rhizosphere metabolomics is the collective chemical fingerprint of the rhizosphere, consisting of root exudates and the compounds produced by rhizosphere organisms. The rhizosphere metabolome is a reservoir of novel antibiotics owing to the millions of unique microorganisms found in soil and enriched in the rhizosphere. Knowledge of the rhizosphere metabolome, its wealth of novel antibiotics, and its role in natural disease suppression has been limited by the fact that soil is the most difficult matrix from which to isolate antibiotics due to its complexity and variation in chemical and physical properties. Methods of sample preparation have been time consuming, and antibiotic extraction efficiency and reproducibility vary from soil to soil. This review focuses on the extraction and quantification of soil antibiotics, the evolution in technology to detect and quantify them, and the use of ultrahigh performance liquid chromatography–time-of-flight mass spectrometry (UHPLC–TOF-MS).