Ganoderma lucidum P. karst is an edible fungus that is used in traditional medicine and contains triterpenoids as the major phytoconstituents. Ganoderic acids are the most abundant triterpenoids that showed pharmacological activity. As Indian varieties contain ganoderic acid H (GA-H), we aimed to prepare GA-H-based triterpenoid enriched fraction (TEF) and evaluated its pharmacokinetics, metabolomics, and stability analysis. A high-performance liquid chromatography (HPLC) method was developed to quantify GA-H in TEF and rat plasma. Based on GA-H content, a stability assessment and pharmacokinetic study of TEF were also performed. After its oral administration to rats, TEF’s the metabolic pattern recognition was performed through ultra-performance liquid chromatography mass spectroscopy (UPLC–MS). The developed HPLC method was found to be simple, sensitive, precise (90% recovery) for the quantification of GA-H. Pharmacokinetic analysis showed that GA-H reached its maximum plasma concentration (C_max 2509.9 ng/mL) within two hours and sustained quantifiable amount up to 12 h with a low elimination rate (K_el) 0.05 L/h. TEF contained ten bioavailable constituents. The prepared TEF was found to be stable for up to one year at room temperature. The prepared TEF, enriched with ganoderic acid, is stable, contains bioavailable constituents, and can be explored as phytopharmaceuticals for different pharmacological properties. Highlights: (1). Preparation of triterpenoid enriched fraction (TEF) from Ganoderma lucidum. (2). Major triterpenoid in TEF is ganoderic acid H (GA-H). (3). TEF contains several bioavailable phytoconstituents. (4). TEF (considering only GA-H) is stable for up to one year at room temperature. (5). GA-H is rapidly absorbed and has high systemic exposure.