While monitoring and control of inoculum characteristics at the seed fermenter stage are well established in most bioprocesses, the very first steps of culture expansion in shake flasks are usually neglected, even though they are influencing all following process steps. Especially for organisms with complex metabolic regulation or morphological variability, the utilization of a suboptimal inoculum can severely worsen the bioprocess outcome. In this application note, we present an integrated approach for optimal inoculum preparation in shake flasks with automated seed inoculation, conditioning and online monitoring, which allows the generation of reproducible inoculums and the flexibility to adjust the harvesting time according to the operator’s requirements.