Background: Cardiovascular diseases with the accompanied elevated level of total cholesterol have been a major problem in society for the last several decades. They belong to the diseases of civilization which affect people at an increasingly young age. For this reason, our aim was to investigate whether the concentrations of selected steroids are related to elevated total cholesterol in people without diagnosed cardiovascular diseases. Material and methods: The study involved 71 plasma samples. 19 of them were obtained from women and men with elevated cholesterol levels, whereas 52 samples were from healthy volunteers (control group). Liquid chromatography coupled with mass spectrometry (LC-MS) validated method followed by solid-phase extraction procedure were applied to measure the plasma concentrations of the three endogenous glucocorticosteroids (cortisol, corticosterone and cortisone). Results: Statistically significant differences between the concentration of cortisol were noted among healthy women and women with elevated cholesterol. The measured concentrations of cortisol in healthy women and men are comparable, 111.19 ng/mL and 112.22 ng/mL. respectively. However, the concentrations of cortisol in the elevated cholesterol group was significantly lower among women with elevated cholesterol than in healthy women (74.13 ng/mL and 111.19 ng/mL respectively). The concentration of cortisol for men with elevated cholesterol was 38.60 ng/mL. Hence, it is much higher than in women with elevated cholesterol and higher than in the case of healthy men. Distinctive changes can be observed also for corticosterone measured for both women and men. Conclusions: The observed differences on the level of steroids between healthy control group and patients with elevated cholesterol can be considered as worthy of further investigation from both biochemical as well as clinical points of view.