To investigate the effect of physicochemical factors on the formation of albumin nanoparticles via desolvation method followed by heat stabilization, Irinotecan (IRT) was chosen as an anticancer model drug. Albumin nanoparticles were prepared by desolvation method using ethanol followed by heat stabilization. Various factors such as pH, temperature, and the amount of desolvation agent were changed in the preparation process. The size, surface charge and the morphology were then determined by dynamic light scattering (DLS) and electrophoretic light scattering spectrophotometer (ELS) and transmission electron microscopy (TEM), respectively. Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectropolarimeter (CD) were used to investigate intermolecular and structural changes of albumin after heat stabilization. Loading efficiency of drug and drug contents of IRT loaded albumin nanoparticles were measured. The particle size range of albumin nanoparticles was in 100-200nm. The particle size increased as heating, lowering pH and increasing the amount of desolvation agent. Surface charges of albumin nanoarticles were negative, indicating the good stability of the nanoparticles via electrostatic repulsion. TEM data showed a uniformly spherical shape of albumin nanoparticles upon various conditions. FT-IR data confirmed the increase of hydrogen bonding after heat stabilization. CD data showed changes in secondary structure of albumin after heat stabilization. IRT was successfully loaded as an amorphous form to the optimal formulations with high encapsulation efficiency of 78.20% and drug loading content of 31.28%. The physicochemical properties of albumin nanoparticles were varied by preparation factor such as pH, temperature, and the amount of desolvation agent. This research offered the optimal conditions and mechanism for the preparation of albumin nanoparticles for further drug delivery.