<p>A highly sensitive, rapid, and straightforward LC–MS/MS technique has been developed and meticulously validated for the detection of prucalopride in human plasma using prucalopride-13CD3 as an internal standard. The thorough validation process, conducted as per the US FDA guidelines, ensures the reliability and accuracy of our method. The analytes were extracted from human plasma via a liquid–liquid extraction technique using methyl tertiary butyl ether. The sample extract was separated on a Kromasil C18 column using a mixture of methanol and 5 mM ammonium formate in 0.1% formic acid (w/v) (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min within a run time of 2.20 min. The eluents were quantified in positive ion mode by multiple reaction monitoring to observe the transitions from m/z 368.0 to 196.0 for prucalopride and from m/z 372.0 to 196.0 for the internal standard. The linearity of the method was established within the range 50–12,000 pg/mL, and the recoveries obtained were 89.92 and 90.42% for the analyte and internal standard respectively. The proposed method can be applied to quantify prucalopride during therapeutic drug monitoring and bioavailability/bioequivalence studies in humans.</p>