Both strain engineering and process optimization are intensively studied in microbial biosurfactant literature. However, screening of multiple strains and/or medium components in parallel is a very labor-intensive and timely process, considering the only applied technique nowadays is evaluation through shake flask and/or bioreactor experiments. Therefore, in this work, the development, optimization, and application of a more throughput techniquebased on 24-deep well platesare described for a new Starmerella bombicola strain producing bolaform sophorolipids. To develop an optimal setup, the influence of plate position and culture volume and the type of sandwich cover was investigated. Optimal parameters, which did not result in significant differences compared with shake flask experiments concerning growth, glucose consumption, and production of novel sophorolipids, were defined and validated. Next, the new method was applied to evaluate the influence of the use of alternative (commercial) nitrogen sources in comparison with the yeast extract currently applied in the production medium, aiming to increase production efficiency. Self-made yeast extracts from S. bombicola cells were also included to evaluate possible recycling of cells after fermentation. In conclusion, the designed method enabled the efficient and successful comparison of ten different nitrogen sources in varying concentrations (1, 4, and 10g/L) on bola sophorolipid production, which can now also be performed for other parameters important for growth and/or glycolipid production.