Pongamia pinnata has been established as a biofuel legume tree. Different methods such as acetylene reduction assay, ureide analysis, isotopic techniques, nitrogen difference method and isotope techniques (¹⁵N natural abundance and ¹⁵N enrichment technique) were employed to analyze, assess and estimate symbiotic nitrogen fixation in 16-wk-old pongamia seedlings. In the acetylene reduction assay, the uninoculated control had negligible ethylene produced while inoculated plants were shown to have increasing ethylene production from 0-min to 60-min incubation with a range of 0.03 to 2.76 mL. Pongamia was found to produce 1.19 x 10^-6 mole per plant with a Rhizobum (PR-UQ-05) inoculation. Ureide analysis was also done, not only to estimate fixed nitrogen, but also to determine the presence of allantoin in the xylem of pongamia. The result showed that allantoin was present in pongamia at low levels of 143–150 nmole, which means pongamia utilizes ureides in the form of allantoin to transport its fixed nitrogen to other plant parts to support growth and reproduction. The nitrogen difference method and isotope techniques quantified the fixed nitrogen of pongamia inoculated with PR-UQ-05 which was estimated to be 100 mg/plant (based on the difference of total N yield between the nodulated and non-nodulated pongamia seedlings) and from 20.4 mg/plant (natural abundance) to 47.4 mg/plant (enriched). The different methods used in this study showed different results based on the amount of fixed nitrogen calculated for each method. However, all of the methods employed in this study demonstrated that Pongamia inoculated with PR-UQ-05 fixed more nitrogen than the uninoculated control. The symbiotic nitrogen fixation of Pongamia demonstrated in this study is very relevant to the biofuel industries.