The adrenocortical human cell line H295R is a valuable tool for screening endocrine disrupting compounds. In general, previous research focus has been on the production of the 2 sex steroids, 17β-estradiol and testosterone, and less attention has been paid to other important steroid end points in the steroidogenesis with a wide range of physiological functions, such as the glucocorticoids (corticosterone and cortisol). A newly developed and validated solid phase extraction (SPE) liquid chromatography-mass spectroscopy (LC-MS/MS) method was used to measure the production of cortisol and corticosterone in the H295R cell line. The method was applied by studying the effects of 2 model endocrine disrupters, ketoconazole and prochloraz, the pharmaceutical budesonide, and the inducer forskolin on the steroid production in this cell line. Dose-response curves were obtained for the correlation between hormone concentrations and the concentration of the individual disruptors. Exposing cells to ketoconazole resulted in a decrease in cortisol and corticosterone concentrations in a dose-dependent manner with EC₅₀ values of 0.24 and 0.40 μmol/L, respectively. The same applied for cells exposed to prochloraz with EC₅₀ values of 0.06 and 0.09 μmol/L for cortisol and corticosterone, respectively. Budesonide also inhibited glucocorticoid secretion. The EC₅₀ value for cortisol was 19.50 μmol/L, whereas the EC₅₀ value for corticosterone was 71.42 μmol/L. Forskolin induced the secretion of both cortisol (EC₅₀ = 4.09 μmol/L) and corticosterone (EC₅₀ = 0.28 μmol/L). The results obtained demonstrated the validity of the method. Based on these findings, quality criteria for the production of these steroids in this cell line were suggested.