Background: Diacerein, a prodrug of Rhein, is commonly prescribed for the management of joint disorders, specifically osteoarthritis. This study aimed to develop and validate an LC-MS/MS method to quantify Rhein and its major metabolites, Rhein-G1 and Rhein-G2, in plasma samples. Method: An ACE C18 column was used for chromatographic separation with a mobile phase comprising ammonium acetate at a concentration of 1.0 mM and acetonitrile. Detection was achieved using a Sciex 4000 Q-Trap LC-MS/MS, operated in negative ion mode with multiple reaction monitoring (MRM). Results: The analytical results indicated that the lower limit of quantification (LLOQ) for Rhein and its glucuronides was 7.81 nM. Precision was consistently below 9.14%, while accuracy remained within the acceptable range of 80.1–104.2%. We also verified the method’s matrix effect recovery and stability variance, which were less than 12.60% and 10.37%, respectively. The pharmacokinetic study demonstrated that diacerein is swiftly metabolized into Rhein, and then Rhein subsequently undergoes glucuronidation, forming detectable concentrations of Rhein-G1 and Rhein-G2 in plasma. Conclusions: This new LC-MS/MS method proved to be both sensitive and selective, allowing for pharmacokinetic studies in rats.