A novel method for the estimation of Budesonide in human plasma by using LC-MS-MS and the analyte is budesonide and internal standard is levenorgestreol were extracted with the tertbutyl methyl ether: n-hexane (70:30, v/v) from human plasma. The chromatographic severance was attained of the peak using Agilent Zorbax Eclipse XDB-C8, (100 mm × 4.6 mm, 3.5 μm) column with a run time is 2.5 min. Budesonide and levenorgestreol were recorded at the total ion current of their relevant multiple reaction monitoring. The LC-MS-MS system composed an Agilent 1100 infinity combined with an AB Sciex Qtrap® 4000 thermo Finnigan TSQ quantum discovery triple quadruple mass spectrometer. All of the parameters must be validated like selectivity, accuracy, precision, linearity, lower limit of quantification, matrix effect, recovery reached the acceptance criteria under the following of ICH guidelines. Budesonide have checked the various stability studies like short term stability at 25°C, long term stability for 55 days at -70°C, wet extract stability for 54 h, auto sampler stability for 63 h, bench top stability for 14 h and freeze-thaw stability at -60°C. Hence, it can be used for routine drug analysis and bioequivalence studies of budesonide in human plasma samples.