The reproducibility of results is an essential prerequiste for a successful systematic analysis of the influence of process development parameters on fermentation performance. Concerns of inoculum inconsistency and strain instability in Xanthan fermentations have led to the use of a highly productive variant of Xanthomonas campestris NRRL-B1459 under strict preservation protocols and the development of an improved, yet simple inoculation strategy. This technique allows the propagation of standardized inocula, ensuring that starter cultures both identical in concentration and history are used. Implementation of this methodology resulted in extremely reproducible cultivations in shake flask and in fermenters of up to a pilot scale of 150 L in volume.