In this work, the influence of the ionic strength and phosphate ions on poly(L-lysine) hydrodynamic radius, conformation and persistence lengths has been studied for molar masses comprised between 3000 and 70 000 g/mol. Mark−Houwink coefficients have been obtained via the determination of poly(L-lysine) hydrodynamic radius using Taylor dispersion analysis. The influence of phosphate ions and ionic strength on the solvent quality (poor, Θ, or good solvent) for poly(L-lysine) have been studied in details. Quantitative data on hydrodynamic radius, persistence length, Mark−Houwink coefficients are provided at pH 7.4, in the range of 10 mM to 1 M ionic strength, and for different phosphate ion concentrations from 0.1 mM to 50 mM under physiological conditions (154 mM ionic strength, pH 7.4). The strong influence of phosphate ions on poly(L-lysine) properties was finally illustrated by studying the interactions (stoichiometry, binding constant, and cooperativity) between poly(L-lysine) of DP 50 and human serum albumin, in the absence and in the presence of phosphate ions at pH 7.4.