This paper describes a method that includes an optimized extraction process and identification and quantification of two anticancer compounds (garcinol and isogarcinol) by LC/electrospray ionization (ESI)-MS/MS in the multiple reaction monitoring (MRM) mode. The study aimed to develop a fast, accurate, and sensitive method for the quantification of garcinol and isogarcinol in different extracts of Garcinia indica fruits. The compounds were detected using LC/ESI-MS/MS in the positive-ion mode and quantified in the MRM mode using a transition mass of m/z 603.3/411 taken as the quantifier and 603.3/343.2 as the qualifier for garcinol and isogarcinol. Five point calibration curves were linear in the range of 2 to 10 ng/mL for garcinol and 0.5 to 6 ng/mL for isogarcinol, with a correlation coefficient of ≥0.990 for both. LOQ for garcinol and isogarcinol was 0.06 and 0.05 ng/mL, respectively, while LOD was 0.021 and 0.017 ng/mL respectively. Our work demonstrated optimization of extraction procedure, fast and highly sensitive quantification (pg level LOQ), and validation of the developed method for the investigated compounds in fruit extracts of G. indica.