Surfactants, even in miniscule amounts, are often used for the synthesis and especially the stabilization of nanomaterials, which is essential for in vivo applications. In this study, we show that the interaction between nanoparticles and proteins strongly depends on the type of stabilizing surfactants and their (small) concentration changes. The reaction between human serum albumin and polystyrene nanoparticles stabilized by an ionic or nonionic surfactant-sodium dodecyl sulfate or Lutensol AT50, respectively-was monitored using isothermal titration calorimetry. It was found that the amount of surfactant molecules on the surface significantly determines the protein binding affinity and adsorption stoichiometry, which is important for all nanomaterials coming into contact with biological components such as blood plasma proteins. Thus after synthesizing nanomaterials for in vivo applications as drug delivery agents, it is crucial to perform a detailed analysis of the obtained surface chemistry that accounts for the presence of minimal amounts of stabilizing agents.