Experiments carried out in our laboratory showed that In(III)-mesotetraphenylporphyrin (InTPP) was a better photosensitizer than Photofrin to photooxidate erythrocytes. However, InTPP is a lipophilic compound, hampering its administration by via intravenous. Thus, we encapsulated the InTPP in poly(DL-lactide-co-glycolide) (PLGA) nanospheres prepared by way of the emulsification/evaporation method to solve this problem. As researchers have reported that particles with smaller diameters than 200 nm are required to avoid spleen filtering effects, we decided to optimize the particle size by way of a 2 ^{4 -1} fractional factorial design (FFD). The particle size was monitored using the integrated area of absorbance spectra (IAAS) for each colloidal suspension. The factors examined were the agitation rate (10,000-14,000 rpm), the percentage of polyvinyl alcohol (PVA) (1-2% w/v) and the percentage of ethanol in the aqueous (0-11% v/v) and organic (0-5% v/v) phases. The results showed that increasing the levels of all factors led to a reduction in the particle size since the IAAS decreased from 562 ± 3 to 213 ± 15. The suspension with the smallest IAAS value was also analyzed by a particle size analyzer, and the average diameter of the spheres was 262 ± 13 nm. PVA and ethanol percentages in aqueous phase were the main factors that influenced in the particle size. Studies have shown that PVA is a non-biodegradable polymer and is capable of causing cancer. Thus, we took a new step by increasing the agitation rate and the percentage of ethanol in the aqueous phase, maintaining the PVA percentage fixed at 1% (to reduce the residual PVA on the spheres) and the ethanol percentage in the organic phase at 5% v/v. The smallest value of the IAAS was 65 ± 1 when the agitation rate and the ethanol percentage used in the particle preparation were 18,000 rpm and 30% v/v, respectively. The average diameter of the spheres in this formulation was 118 nm and the results from the scanning electron microscopy matched the particles' size. The FFD results suggest that it is possible to optimize the particle size by increasing the agitation rate and the percentage of ethanol  in the aqueous phase, and by using the IAAS of colloidal suspension to monitor the particle size. Supported by Unicamp (01-P-10228-2004) and FAPESP (2003/09536-9).