Introduction: Prolactin (PRL) is a protein hormone. It is excreted by adenohypophysis and other tissues to a lesser extent. Its production is primarily stimulated by estrogens while secretion is under the control of prolactin inhibiting factor (PIF) - dopamine. Its structure is similar to growth hormone (hGH) and human placental lactogen (hPL). In healthy patients serum prolactin appears in three forms of different relative molecular mass: prokaryotic monomer (23kDa) 86%, large prolactin (50-60 kDa) 9% and large-prolactin (> 150 kDa) 5%. Concentration of prolactin in serum of women is significantly higher than in men due to estrogen influence. Some physiological conditions such as sleep, physical exertion, stress, hypoglycemia, pregnancy and lactation are also elevated. The most common pathological conditions associated with prolactin are hyperprolactinemia caused by prolactinomas, a benign pituitary tumor. Aim: The aim of this paper is to prove the absence or presence of statisticaly significant differences in the results of serum prolactin determined by two different methods. Materials and Methods: A sample of 40 randomized patient samples from ambulatory patients was tested. Two different immunochemical methods were compared: the immunoradiometric method (IRMA) and the electrochemiluminiscence immunoassay method (ECLIA). Results: High coefficient of correlation (r = 0.916) and significance level P <0.0001 were obtained. The confidence interval (95% CI) for coefficient of correlation was 0.845 to 0.955. Data obtained by using the Passing and Bablock regression analysis showed that there are no constant deviations between methods but there is a slight proportional deviation. Cusum's test does not show significant linear deviations (P = 1.00). Data obtained using Deming regression analysis showed that there were no constant or proportional deviations between methods. Conclusion: Monitoring of the value of prolactin in each patient is important with the application of the same methods on the same analyzer to make the results comparable. If prolactin is determined by other methods it is necessary to determine the new zero concentration of prolactin from which this hormone concentration can be monitored.