Streptomycesspecies are intensively studied for their ability to produce a vari-ety of natural products. However, conditions influencing and leading to prod-uct formation are often not completely recognized. Therefore, in this study,high-throughput online monitoring is presented as a powerful tool to gain in-depth understanding of the cultivation of the model organismStreptomycescoeli-colorA3(2). Through online measurements of oxygen transfer rate and autofluo-rescence, valuable information about availability of nutrients and product for-mation patterns of the pigments actinorhodin and undecylprodigiosin can beobtained and explained. Therefore, it is possible to determine the onset of pig-mentation and to study in detail the influencing factors thereof. One factor iden-tified in this study is the filling volume of the cultivation vessel. Slight variationsled to varying pigmentation levels. By combining optical and metabolic onlinemonitoring techniques, the correlation of the filling volume with pigmentationcould be explained as a result of different growth trajectories caused by varyingspecific power inputs and their influence on the pellet formation of the filamen-tous system. Finally, experiments with the addition of supernatant from unpig-mentedandpigmentedculturescouldhighlighttheapplicabilityofthepresentedapproach to study quorum sensing and cell-cell interaction.