Nanoparticles prepared of human serum albumin (HSA) represent promising carriers for drug delivery. Desolvating procedure is a common method to obtain protein-based particles. However process parameters have a great influence on size, size distribution and particle yield of the resulting formulation. Therefore, it is crucial to investigate their effects on particle preparation. This study is focused on the influence of different desolvating agents on the resulting particle characteristics and cytotoxicity in cell culture. Another aspect of the study was the influence of different stirring rates, pre-stirring of the HSA solution and the continuous addition of desolvating agent during the preparation process. The results indicate that it is possible to predict the particle size depending on dielectric constant of the desolvation medium. Remarkably, combination of methanol and ethanol used as desolvating agent was able to produce very small spherical HSA nanoparticles in a size range between 50 and 80 nm.