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A simple sensitive and accessible ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry using AJS-ESI method is established and validated for the intended purpose of quantification of major cholesterol steroidal metabolites Corticosterone, 11α-Hydroxyprogesterone, Testosterone and Progesterone is extracted by Tertiary butyl methyl ether solvent from human plasma and analyzed using Infinity Lab Poroshell 120 EC-C18, (2.1 × 150 mm, 2.7 µm) column by gradient elution. The Eluent composed of 5 milli molar Ammonium formate in Methanol as eluent A and 5 mM Ammonium formate as eluent B and detection was done by AJS- ESI fitted tandem mass spectrometry in positive mode. The internal standard used is Caffeine. The quantification lower limits (LLOQ) are 0.200 ng per liter for all four steroids Corticosterone, 11α-Hydroxyprogesterone, Testosterone and Progesterone. The Calibration curve are linear over the respectively limits of 0.200 to 1000.000 ng per milliliter of Plasma for each analyte and quadric for Testosterone. This new LC-MS/MS method shows presentable precision & accuracy and provides satisfactory sensitive for the fulfillment of human pharmacokinetic studies.