Proteins from various sources are widely used in the food industry due to their unique functional performances in food products. The functional properties of proteins are somehow dictated by their molecular characteristics, but the exact relationship is not fully understood. This review gives a tangible overview of the methods currently available for determining protein functionality and related molecular characteristics in order to support further research on protein ingredients. The measurements of protein functionality include solubility, water holding capacity, oil holding capacity, emulsion property, foam property, and gelation. This review also provides a description of different methods of molecular characteristics including electrophoresis, surface hydrophobicity and charge, molecular interaction, and thermal property measurement. Additionally, we have put significant emphasis on spectroscopic methods (ultraviolet-visible, Fourier transform infrared, Raman, circular dichroism, fluorescence and nuclear magnetic resonance). In conclusion, first and foremost, there is a need to agree on a standardization of the analytical methods for assessing functional properties. Moreover, it is mandatory to couple different analyses of molecular characteristics to measure and monitor the structural changes obtained by different processing methods in order to gain knowledge about the relationship with functionality. Ideally, a toolbox of protein analytical methods to measure molecular characteristics and functionality should be established to be used in a strategic design of protein ingredients.