Background: The ICH guidelines Q8 (Analytical Quality by Design (AQbD) technique) give the procedure for optimization of analytical parameter involved method development and validation of Sirolimus (SL) in spiked Plasma and in dosage form by using advanced analytical technique. Objective: This research paper is based on Quality by Design (QbD) finalized conditions for a method for the determination of concentration of Sirolimus by using Liquid Chromatography–Tandem Mass Spectrometry (LC-ESI/MS). Method: Sirolimus is a potent immunosuppresant drug. Critical Process Attributes (CPA) is considered to be an influential parameter in separation, identification and quantification processes by UHPLC-ESIMS/ MS which are pH modifier,organic content, buffer strength, flow rate, Ionization chamber temperature, sheath gas, spray voltage and auxiliary gas that alters Critical Analytical Attributes (CAA), like peak area and Retention Time (Rt). These factors were evaluated first in a factorial design (TAGUCHI) and then extensively in a Central Composite Design (CCD) to zero-in on the mobile phase for the quantification of Sirolimus (SL) standard drug and along with its internal standard ( SL IS) in spiked plasma samples and in the formulation. Pareto chart from initial factorial design (Taguchi) model suggested for which of the CPA factors be given the importance that is to be exhaustively analyzed in the CCD and response surface analysis. An UHPLC instrument with the octadecylsilica column (C18 ) of dimensions (5 µm, 2.1 × 50 mm) was used and selectivity of the column was modified using methanol: water (65:35) methanol as an organic modifier as the mobile phase at a flow rate of 0.6 mL min-1. While spray voltage and Ionization chamber temperature for the method is maintained at the level predicted by the response analysis. Detection was performed using Triple quadrupole MS/MS by multiple-reaction monitoring via a positive electrospray ionization source. The ICH guidelines had elaborated about the parameters to be studied in method validation, i.e., selectivity, linearity, accuracy, precision repeatability system-suitability tests, method robustness,ruggedness, sensitivity and stability were accomplished. Results: The results are very clearly indicated that linearity with r value = 0.9980 in the concentration range of 10–500.0 ng mL-1, an intra- and inter-assay precision of 2.4 and 4.1%, respectively, and recovery studies were found to be between 100.8 and 105.6%. The lower limit of quantification was 0.086 ng/mL in 50 µL of human plasma sample. Conclusion: The present method gives a robust QbD-compliant quantitative UHPLC –ESI-MS/MS method for Sirolimus drug containing plasma samples (spiked). Keywords: AnalyticalQbD, sirolimus, LC-ESI/MS, response surface methodology, CCD technique, validation.