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Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal cortex. Whilst surgery is the preferred treatment, adjunctive therapy with mitotane may be offered post-surgically to minimise the risk of recurrence or, in the absence of surgery, to attenuate progression. Aim: The objective was to evaluate the effects of mitotane treatment on serum protein concentrations in patients treated for ACC with mitotane therapy and compare this to patients with other adrenal neoplasms and a normal pregnant cohort. Methods: Serum cortisol, thyroid function tests, adrenocorticotrophic hormone (ACTH), cortisol-binding globulin (CBG), thyroxine-binding globulin (TBG), gonadotrophins and androgens were measured on plasma and serum samples. Thirty-five patients with ACC were included, and mitotane levels were noted to be sub-/supra-therapeutic. Data were tested for normality, reported as mean ± s.d., and compared to other two cohorts using paired-sample t-test with a 5% P-value for significance and a 95% CI. Results: Patients on mitotane therapy had a higher mean serum CBG concentration compared to the adrenal neoplasm group (sub-therapeutic: 79.5 (95% CI: 33.6, 125.4 nmol/L), therapeutic: 85.3 (95% CI: 37.1–133.6 nmol/L), supra-therapeutic: 75.7 (95% CI: −19.3, 170.6 nmol/L) and adrenal neoplasm: 25.5 (95% CI: 17.5, 33.5 nmol/L). Negative correlations between serum cortisol and CBG concentration were demonstrated within the supra-therapeutic plasma mitotane and adrenal neoplasm groups. Conclusion: Patients with ACC and therapeutic plasma mitotane concentrations had higher serum CBG concentrations compared to those with adrenal neoplasms or pregnant women, and higher serum cortisol. Whilst there was no direct correlation with cortisol and mitotane level, the negative correlation of cortisol with CBG may suggest that the direct effect of mitotane in increasing cortisol may also reflect that mitotane has a direct adrenolytic effect.