In this work, the popular CAS assay for siderophore detection, based on the utilization of chrome azurol S, was redesigned and optimized to produce a new, fast, non-toxic, and easy method to determine a wide variety of microorganisms capable of siderophore production on a solid medium. Furthermore, this specific bioassay allows for the identification of more than one single siderophore-producing microorganism at the same time, using an overlay technique in which a modified CAS medium is cast upon culture agar plates (thus its name "O-CAS", for overlaid CAS). Detection was optimized through adjustments to the medium's composition and a quantifying strategy. Specificity of the bioassay was tested on microorganisms known for siderophore production. As a result, a total of 48 microorganisms were isolated from three different types of samples (fresh water, salt water, and alkaline soil), of which 36 were determined as siderophore producers. The compounds identified through this method belonged to both hydroxamate and catechol-types, previously reported to cause color change of the CAS medium from blue to orange and purple, respectively. Some isolated microorganisms, however, caused a color change that differed from previous descriptions.