Context: Measurement of plasma cortisol by immunoassay after ACTH 1-24 stimulation is used to assess the hypothalamic-pituitary-adrenal (HPA) axis. Liquid chromatography-tandem mass spectrometry (LCMS) has greater analytical specificity than immunoassay and equilibrium dialysis allows measurement of free plasma cortisol. Objective: We investigated the use of measuring total and free plasma cortisol by LCMS and total cortisol by immunoassay during an ACTH1-24 stimulation test to define HPA status in pituitary patients. Design and Setting: This was a case control study conducted in a clinical research facility. Participants: Westudied 60 controls and 21 patients with pituitary disease in whom HPA sufficiency (n = 8) or deficiency (n = 13) had been previously defined. Intervention: Participants underwent 1 μg ACTH1-24 intravenous and 250 μg ACTH 1-24 intramuscular ACTH1-24 stimulation tests. Main Outcome Measures: Concordance of ACTH1-24-stimulated total and free plasma cortisol with previous HPA assessment. Results: Total cortisol was 12% lower when measured by immunoassay than by LCMS. Female sex and older age were positively correlated with ACTH1-24-stimulated total and free cortisol, respectively. Measurements of total cortisol by immunoassay and LCMS and free cortisol 30 minutes after 1 μg and 30 and 60 minutes after 250 μg ACTH1-24 were concordant with previous HPA axis assessment in most pituitary patients. However, free cortisol had greater separation from the diagnostic cutoff than total cortisol. Conclusions: Categorization of HPA status by immunoassay and LCMS after ACTH1-24 stimulation was concordant with previous assessment in most pituitary patients. Free cortisol may have greater clinical use in patients near the diagnostic threshold.