Introduction Every new method that is introduced into the laboratory should be previously verified, that is, it should be confirmed whether the features of the method correspond to the manufacturer's specifications. Objective The aim of the research is to verify the reagent for determining the concentration of estradiol by chemiluminescent immunoassay on the Unicel DxI 600 analyzer by testing the precision, truthfulness, and linearity of the method, to make a comparison with the routinely used method on Alinity i analyzer, and to verify the reference intervals. Materials and Methods To check the precision and truthfulness, commercial control samples were used in two concentration levels and were analyzed for 5 consecutive days. To assess the linearity of the method, commercial estradiol calibrators measured at 5 points in duplicate were used. The comparability of the two methods was tested using Bland-Altman and Passing-Bablok regression analysis. Serum samples from 123 patients were used to test the comparability of the methods. Verification of reference intervals was made using 20 samples. Results Precision is expressed using the coefficient of variation. For the low concentration level, the measured KV was 3.56 % (repeatability) and 3.30 % (interprecision), and for the high one 1.87 % (reproducibility) and 1.85 % (interprecision). The trueness is expressed by bias, - 5.37 for low and - 7.74 for high concentration level. The Wilcoxon test was used for linearity with a significance level of P = 0.625 for the first and P = 0.144 for the second measurement. On the Bland-Altman plot, the two results are scattered beyond the limits of the standard deviations. In Passing-Bablok regression analysis, the confidence interval for the intercept is 0.151 and for the slope of the line is 1.027. Conclusion Acceptable research results were obtained for all examined features. There is no statistically significant difference in the comparison of methods that allow the use of both methods in routine work.