The discovery and development of antibiotics for the treatment of bacterial infections is one of the biggest achievements in modern medicine. However, due to multi-resistant pathogens, the spread and emergence of antibiotic resistance pose a threat to public health around the globe, especially in upper- and middle-income countries. The metabolomics approach is valuable in screening novel antibiotics from natural sources, as well as identifying the target points in bacterial metabolism. Mass-spectrometry and NMR-based metabolomics approaches are successful approaches for the discovery of lead compounds. This reviews highlights the application of the metabolomics in drug discovery and development. Different strategies such as, pathway analysis, classification strategy or integration of genomics, transcriptomics and metabolomics are done for the determination of antibiotic modes of action. However, comparative metabolomics and integrative approach enable the study of the bacterial mechanism of resistance. We believe that metabolomics approaches are the most powerful techniques for the diagnosis of infectious diseases and development of novel antibiotics. Despite the high price and complexity in analyzing the product, the key for portability and boost robustness of metabolomics approaches is the upgradation of separation techniques. Therefore, the use of metabolomics approach in the development of novel antibiotics is an interesting alternative for the discovery of new antibiotics as well as for the improvement of known compounds.