Metabolomics research aims to uncover the complex biochemical pathways involved in biological processes, but the interpretation of metabolites, which play diverse roles within biological systems, remains a significant challenge. In this study, we utilised pathway analysis modules, such as Kyoto Encyclopedia of Genes and Genomes (KEGG) Mapper and MetaboAnalyst to facilitate the visualisation and interpretation of Ganoderma boninense metabolomics data, derived from the gas chromatography/quadrupole-timeof- flight (GC/Q-TOF) mass spectrometry-based experiments. Our analysis revealed a time-dependent classification of 39 identified extracellular metabolites from the methanolic extract of G. boninense, where several metabolic pathways, i.e. starch and sucrose metabolism, galactose metabolism, valine, leucine and isoleucine degradation and citrate (TCA) cycles were found significantly enriched in G. boninense. The integration of pathway analysis tools enabled enhanced biological interpretation, contributing to a deeper understanding of temporal primary metabolic pathways linked to the G. boninense developmental process, energy production and cellular functions over time. These findings underscore the importance of the pathway analysis tools in metabolomics, helping to reveal the biological insights which are hidden within the complex metabolite profiles and thus advancing our understanding of the G. boninense developmental process in vitro.