Human breast milk contains substances with anti-infective roles, immunoglobulins, cells involved in immune responses, prebiotics, and important nutrients for infants. Immunonephelometry, polyacrylamide gel electrophoresis, ultrasound spectroscopy can be used to analyze the composition of human milk. In our research, we have examined the sensitivity of these methods in the quantification of lactose, fat and pH of human breast milk and the sensitivity regarding evaluation of proteins, immunoglobulins (A, G, M) concentrations in both human breast milk (52 samples) and milk powder (48 samples). The results showed the appropriateness of using the ultrasonic spectroscopy only for the analysis of the fat content, pH and lactose. For milk samples immunogram (breast milk and milk powder) immunonephelometry can be considered as a reference method. Polyacrylamide gel electrophoresis remains the method of choice in assessing protein concentration. Breast fed infants have superior immune protection in particular by IgA titers and β-globulins.