Measuring hair cortisol seems to be a good alternative to laboratory tests used thus far in routine endocrine diagnostics, primarily because it is independent of the circadian rhythm of cortisol. Due to the average hair growth of 1 cm per month, the results are related to the average blood cortisol levels over the previous weeks, months or years (depending on the length of the hair sample). The aim of this study is an attempt to apply hair cortisol concentration (HCC) measurements to clinical endocrine diagnostics, based on reference cortisol concentrations in the blood in a population without disorders of the hypothalamic-pituitary-adrenal axis (HPA). In the final selection process, 44 patients were enrolled in the study, all with negative interviews regarding disorders of the HPA and with reference levels of cortisol concentration obtained in routine laboratory tests. In the pre-analytic phase, we used 1 cm proximal hair strands cut from the posterior vertex area of the head, followed by the incubation of a 20 mg hair sample in methanol. The final cortisol measurement was done using an enzyme-linked immunosorbent assay (ELISA). The results of HCC ranged from 2 pg/mg up to 51.63 pg/mg. The diurnal decrease in cortisol levels was significantly lower in females than in males (p = 0.031), but we do not consider that difference to be clinically significant. The difference in the HCC between males and females was not statistically significant (p = 0.767). The linear regression coefficient for age was not statistically significant (p = 0.847). Neither the regression coefficients for gender nor the gender and age interactions were statistically significant (p = 0.815). Hair cortisol concentration measurement, unlike other endocrinological tests, gives information about the cortisol concentration in the long-term perspective. The results obtained in this study may be used as a reference for further research aimed at determining normal values of HCC.