ABSTRACT: BACKGROUND: For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes. RESULTS: To address these issues, we devised a robust, small-scale cultivation platform in the form of MTPs (24-square deepwell) for the filamentous bacterium Streptomyces coelicolor and compared its performance to that of shake flasks and bench-scale reactors. We observed that re-designing of medium and inoculum preparation recipes resulted in improved reproducibility. Process turnaround time was significantly reduced due to the reduction in number of unit operations from inoculum to cultivation. The incorporation of glass beads (ø 3 mm) in MTPs not only improved the process performance in terms of improved oxygen transfer improving secondary metabolite production, but also helped to transform morphology from pellet to disperse, resulting in enhanced reproducibility. Addition of MOPS into the medium resulted in pH maintenance above 6.50, a crucial parameter towards reproducibility. Moreover, the entire trajectory of the process was analyzed for compatibility with bench-scale reactors. The MTP cultivations were found to behave similar to bench-scale in terms of growth rate, productivity and substrate uptake rate and so was the onset of antibiotic synthesis. Shake flask cultivations however, showed discrepancy with respect to morphology and had considerably reduced volumetric production rates of antibiotics. CONCLUSION: We observed good agreement of the physiological data obtained in the developed MTP platform with bench-scale. Hence, the described MTP-based screening platform has a high potential for investigation of secondary metabolite biosynthesis in Streptomycetes and other filamentous bacteria and the use may significantly reduce the workload and costs.