Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5–800 ng/mL for each methadone enantiomer with satisfactory relative bias (−1.0 to 1.0%), repeatability (0.9–4.9%) and intermediate precision (1.4–12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the ±15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.