The development of new microbial growth and analytical techniques is becoming increasingly relevant in relation to ‘unculturable’ organisms. This may involve the modification of existing methods or the development of new, custom procedures. One important application of this is in the symbiotic bacteria of insects. Symbionts, due to adaptations to their host, are often difficult to culture in vitro. With the growing interest in the use of modified microbiomes to control vector-borne diseases, improved culture techniques that further the understanding of an insect’s microbiome are becoming increasingly important. The tsetse fly, genus Glossina, is the insect vector for Trypanosoma brucei. This parasite is responsible for human African trypanosomiasis (HAT), endemic in sub-Saharan Africa, as well as the wasting disease nagana in cattle. The tsetse’s secondary symbiont, Sodalis glossinidius, provides a unique potential target for reducing the spread of T. brucei. Here, we describe the use of metabolic modelling to design an entirely defined growth medium for S. glossinidius. This medium was used to verify predictions about carbon and nitrogen usage in the symbiont, including amino acid and vitamin auxotrophies. Furthermore, we discuss the use of multiobjective evolutionary algorithms combined with flux balance analysis to investigate computationally the evolution of symbioses, with S. glossinidius and its free-living relative S. praecaptivus as an exemplar. This work not only improves our understanding of the metabolic interactions within the tsetse microbiome, but serves also as a template for future investigations into symbiont evolution.