Control of milk urea concentration (MUC) can be used in diagnosis of the energy–nitrogen metabolism of cows. There are more analytical methods for MUC estimation and there are also discussions about their result reliability. Aim of this work was to obtain information for MUC result reliability improvement. MUC and MUN (milk urea nitrogen) were investigated in 5 milk sample sets and in 7 calibration/comparison experiments. The positions of reference and indirect methods were changed in experiments. There were following analytical methods for MUC or MUN (in mg.100 ml−1): – photometric method (PH, as reference) based on paradimethylaminobenzaldehyde reaction; – method Ureakvant (UK, as reference) based on difference measurement of the electrical conductivity change during ureolysis; – method Chemspec (CH) based on photometrical measurement of ammonia concentration after ureolysis (as reference); – spectroscopic method in mid infrared range of spectrum (FT–MIR; indirect routine method). In all methodical combinations the correlation coefficients (r) varied from 0.8803 to 0.9943 (P < 0.001). In this way all relationships were relevant. The limits of accuracy and precision of FT–MIR are depend on reference method. Therefore, to pay attention to reference methods results is recommended. Both UK and PH could be calibrated to each other with similar parameters. The MUC reference methods (UK and PH) had better values (lower standard deviations) of accuracy (from 1.14 to 1.81 mg.100 ml−1) and comparable values of repeatability (from 0.65 to 1.83 mg.100 ml−1) as compared to FT–MIR MUC or MUN methods (from 1.39 to 5.6 and from 0.76 to 1.92 mg.100 ml−1) in performed experiments.