Zeaxanthin is a powerful antioxidant that is widely found in vegetables and fruits. Epidemiological evidences suggest that increasing the consumption of zeaxanthin in the diet is associated with a lower risk of age-related macular degeneration, helps prevent glaucoma and cataracts, and supports normal eye health. Zeaxanthin is a promising nutraceutical with many applications in the feed, food, and pharmaceutical industries. Currently, the commercial production of zeaxanthin is still dependant on synthetic routes with limitation for the biological one. Nevertheless, the biotechnological production of zeaxanthin is emerging due to its safety, potential large-scale production, and consumers’ demand and preference for natural additives. Using a rapid screening method based on 16S rRNA gene and effective high-performance liquid chromatography (HPLC)-Diodearray-MS methods for carotenoids’ analysis, we isolated effective zeaxanthin-producing bacteria (strain TDMA-5T and -16T ) that belong to the family Sphingobacteriaceae and Sphingomonadaceae , respectively. In this chapter, we provide a detailed description of the HPLC-Diodearray-MS methods used for rapid analysis and identification of the carotenoids produced by both strains. In addition, the polyphasic taxonomic analysis of both novel strains and the description of a novel species and genus are described.