<p>5-Methylcoumarin-4β-glucoside (5-MC4βG) is coumarine compound isolated from <i>Vernonia glaberrima</i>. The compound has shown potentials as a chemotherapeutic agent for colorectal cancer <i>in vitro</i> and <i>in vivo</i>. To further its preclinical development, a sensitive and robust analytical method for 5-MC4βG quantification is essential for its pharmacokinetic characterization and potential therapeutic application in colorectal cancer. This study presents a validated HPLC method with UV detection for the quantification of 5-MC4βG in rat plasma, urine, and faecal matter. The method demonstrated high linearity (<i>r</i>² &gt; 0.995), low limits of detection and quantification, and acceptable precision, accuracy, and robustness in all three biological matrices. Using this method, the pharmacokinetic analysis of 5-MC4βG was conducted following oral administration. The pharmacokinetic profile revealed a biphasic absorption pattern with two distinct plasma concentration peaks at 1 h and 16 h, suggesting enterohepatic recirculation or dual-phase absorption. The maximum plasma concentration of 0.325 μg/mL and a time to peak concentration of 1 h indicate rapid initial absorption. Excretion studies revealed renal clearance of 5-MC4βG through urine within 1 h, while faecal excretion peaked at 8 h, highlighting the role of both renal and biliary clearance pathways. The presence of 5-MC4βG in the small intestine and colon at 24 h and 48 h further supports enterohepatic recirculation. Prolonged exposure and site-specific accumulation in the colon may improve therapeutic efficacy against colorectal tumors. This study establishes a foundation for further preclinical investigations of 5-MC4βG as a chemotherapeutic agent for colorectal cancer. </p>