Rottlerin is a key bioactive phytoconstituent present in the pericarp of . It shows promising multifaceted pharmacological actions against cancer. However, there is hardly any report for the quantification of rottlerin in the biological matrix and on its pharmacokinetic behavior. Therefore, we aimed in the present study to assess selective ADME properties and pharmacokinetics of isolated and characterized rottlerin using a newly developed and validated liquid chromatography-tandem mass spectrometry-based highly sensitive bioanalytical method. The method was found to be simple (mobile phase and analytical column), sensitive (1.9 ng/mL), and rapid (run time of 2.5 min). All the validation parameters were within the acceptable criteria of the United States Food and Drug Administration's bioanalytical method validation guideline. The method was found to be very useful to assess lipophilicity, plasma stability, metabolic stability, plasma protein binding of rottlerin, as well as its oral and intravenous pharmacokinetics in mice. Rottlerin showed a number of drug-like pharmacokinetic properties (). Moreover, it displayed an excellent half-life (>2 h) and oral bioavailability (>35%) as compared to other members of natural phenolics. The present study is the first-time report of ADME properties and preclinical pharmacokinetics of rottlerin. The generated information is very much useful for its further development as a phytotherapeutics toward cancer therapy.