We describe an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3min. The limit of quantitation for cortisol and cortisone in plasma was 3.75nmol/L and linearity extended to 2000nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5nmol/L and for dexamethasone 1nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC=0.79×IA+31.12 with R(2)=0.960 (p<0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC=1.06×HPLC+9.82, R(2)=0.992 (p<0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.