The development of a new vaccine requires the precise characterization of all the physicochemical parameters of the vaccine antigens, which are the molecules that induce the immune response. Taylor dispersion analysis (TDA) is a promising alternative technique for the determination of diffusion coefficients and hydrodynamic radii of proteins, macromolecules and nanoparticles. In this work, TDA was used to determine the hydrodynamic radius distribution and its average value of four antigens: diphtheria toxoid (DT), tetanus toxoid (TT), hepatitis B surface antigen (HBsAg) and polyribosyl-ribitol phosphate conjugated to tetanus toxoid (PRP-T). The robustness of the results obtained was investigated on bare fused silica capillary and hydroxypropylcellulose coated capillary. The impact of operational parameters on the limit of detection (LOD) and limit of quantification (LOQ) were studied from both theoretical and experimental points of view. The influence of the diameter and the length of the capillary on the LOD and LOQ were studied as well as the impact of the mobilization pressure. General guidelines for the choice of the initial operating conditions are given for the development of future TDA methods.