The CP4 EPSPS gene is widely used in herbicide-tolerant crops/plants all over the world. In this study, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify the amount of CP4 EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was used to measure the unique peptides of the CP4 EPSPS protein. Two peptides unique to CP4 EPSPS were synthesized and labelled in H₂¹⁸O to give ¹⁸O stable isotope labelled peptides which served as internal standards. The validated method resulted in good specificity and linearity. The intra- and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4 EPSPS in the crude extract without time-consuming pre-separation or purification procedures.