Hemophilia B is a rare X-linked hereditary blood clotting disorder caused by a complete or partialy deficiency of coagulation factor IX activity that results with increased bleeding tendency. Since residual FIX activity correlates well with the clinical picture in most cases, hemophilia B patients are classified as severe, moderate or mild based on FIX activity measured by the one-stage coagulation method. With regard to the increasing use of the chromogenic method in the diagnosis of hemophilia and its advantages compared to the standard coagulation method, the goal of this investigation was to examine the influence of the determination of FIX activity by the chromogenic method on the classification of hemophilia B patients and to examine the differences in the results of the therapeutic monitoring by coagulation and chromogenic methods in order to determine which method is more suitable for monitoring the effectiveness of drugs and thereby contributes to better care and treatment of hemophilia B patients. The investigation has included 45 male subjects (33 adults and 12 pediatric) suffering from hemophilia B, of whom five were receiving replacement therapy at the time of the examination, and they were obsereved as a separate group during the statistical analysis of results. APTT and FIX activities were measured by using the coagulation method (FIXkgl) and chromogenic method (FIXkr) using the BIOPHENTM FIX commercial test (HYPHEN BioMed, Neuville sur Oise, France) in all subjects on the Atellica COAG 360 analyzer (Siemens Healthcare, Marburg, Germany). In relation to FIXkr activities, FIXkgl activities were significantly higher in mild and moderate hemophilia B patients with the exception of the severe form where higher FIXkr values were observed. The most discrepancies in classification with both methods were observed in severe and moderate hemophilia B forms, while the least discrepancies in classification were observed in mild forms of hemophilia. Moreover, a better correlation of the clinical phenotype with the classification according to the chromogenic method was observed. In the group of patients on replacement therapy, statistically significant difference was not found between the activities of FIXkr compared to FIXkgl activities. The largest discrepancies between the results of FIXkr and FIXkgl in the group of patients on replacement therapy were observed in the area of higher FIX activities. The results of this investigation indicated the complexity of the differential-diagnostic approach in the diagnosis of hemophilia B and the indispensable role of the coagulation and chromogenic methods in the diagnosis and monitoring of replacement therapy. Also, the advantages and disadvantages of each method were observed, and the basis for the potential application of the chromogenic method in the classification and therapeutic monitoring of patients with hemophilia B was provided.