Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.