Ganoderma lucidum (Lingzhi), a medicinal mushroom, is recognized for its broad pharmacological activities, including potential health and longevity benefits. Notably, several triterpenoids derived from its spores and fruiting body have demonstrated anti-inflammatory, anti-tumor, and immunomodulatory effects. Despite their therapeutic promise, pharmacokinetic parameters and tissue distribution profiles of these bioactive components are not well characterized, representing a significant knowledge gap. This study aims to elucidate the pharmacokinetic characteristics and tissue distribution patterns of major triterpenoids in sporoderm-removed Ganoderma lucidum spore powder (RGLSP) in rats, thereby laying the groundwork for further optimization of these natural products. We established an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS) method to quantify 12 triterpenoids in rat plasma and tissues following oral administration of RGLSP. The method was validated according to US Food and Drug Administration bioanalytical guidelines and demonstrated good performance. Pharmacokinetic analysis revealed that the mean time to peak concentration for the 12 triterpenoids ranged from 0.25 to 2.33 h, with maximum concentrations varying from 42.52 to 643.13 ng/mL and the area under the concentration-time curve spanning 72.74 to 943.00 ng·h/mL. Tissue distribution results indicated rapid and extensive distribution of the triterpenoids in rat liver, lung, spleen, kidney, heart, and gastrointestinal tract, followed by gradual metabolism. After oral administration, major triterpenoids in RGLSP were rapidly absorbed into the plasma and widely distributed across five major viscera and the gastrointestinal tract, undergoing hepatic and intestinal circulation through metabolic pathways. These findings provide a valuable reference for the clinical application of RGLSP, as well as lead optimization of the triterpenoids.