Ganoderma lucidum is a rich and promising source of bioactive compounds capable of positively influencing the human health. For this reason, G. lucidum is the target of vigorous investigational activities, focusing on the extraction of substances with potential biomedical applications, as well as on the improvement of analytical methodologies for their screening. In the present study, three different extraction processes (i.e., extraction with n-hexane, in Soxhlet apparatus with n-hexane and reflux with chloroform) were employed to evaluate the lipid content of G. lucidum spores. The experimental data from the produced extracts were compared with ones from the commercial liquid extract obtained with supercritical CO2 extraction of G. lucidum spores. To achieve this goal, the gas chromatography-mass spectrometry (GC-MS) methodology was optimized for an accurate and reliable identification and quantification of fatty acids in triglycerides of G. lucidum spores. Significant amounts of oleic and palmitic acids were found in the examined extracts. In particular, our findings show that economical and convenient extraction procedures, which are simple, low cost and no need for expensive tools, allow to produce extracts with a lipid composition similar to the one obtained with hard strategies of extraction.