Nontypeable Haemophilus influenzae (NTHi) is a human-host adapted pathogens that causes mucosal infections including otitis media, pneumonia and exacerbates chronic respiratory diseases such as chronic obstructive pulmonary disease and asthma. Metabolic adaptation of pathogens to the host is emerging as a key aspect of intracellular persistence, recurrence, and virulence. A recent study in our laboratory demonstrated NTHi relies on organic acids such as lactate, nucleosides, pentose, and triose sugars for growth, and that L-lactate utilization via L-lactate dehydrogenase (LldD) was required for Hi long-term persistence into the host cells (Hosmer et al. 2022). However, the role of the other substrate groups for NTHi virulence has not been studied yet. The current study aimed to address this gap in knowledge and evaluate key enzymes that could be a potential target for drug design. Growth of Hi2019 wild-type (WT) strain (COPD isolate) was assayed using chemically defined medium (CDM) with varying concentrations of different substrates including lactate, ribose, deoxyribose, N-acetyl glucosamine, α-hydroxybutyrate, dihydroxyacetone, and uridine, confirming that Hi2019 can use all these substrates as carbon sources. To better understand the significance of pentose utilization, Hi2019 strains with a single gene mutation in ribose uptake (rbsB) and phosphorylation (rbsK) were constructed, and initial data indicates an inability to grow on ribose and deoxyribose-containing medium, but wild-type growth when glucose was used as the carbon source. Following this phenotypic validation, we are currently evaluating the effect of rbsB and rbsK mutations for NTHi virulence. Future work could include enzymatic characterization of these proteins, similar to work that is already underway for the LldD. We have successfully overexpressed this enzyme with N- or C-terminal polyhistidine tags in Escherichia coli which enables kinetic characterization and evaluation of potential inhibitors. The full evaluation of NTHi energy generation and its role during host-pathogen interactions is an understudied aspect of NTHi biology and will significantly expand our knowledge of the pathogenesis of this bacterium.