The objective of this study was to develop biodegradable poly(DL-lactide-co-glycolic acid) (PLGA) nanoparticles simultaneously loaded with indocyanine green (ICG) and doxorubicin (DOX). The modified oil in water single emulsion solvent evaporation method was used. To enhance the incorporation of both agents and control particle size, four independent processing parameters including amount of polymer, initial ICG content, initial DOX content, and concentration of poly-vinyl alcohol (PVA) were investigated. The ICG and DOX entrapment in nanoparticles as well as the nanoparticle size were determined. The nanoparticles produced by standardized formulation were in the range of 171+/-2 nm (n=3) with low polydispersity index (0.040+/-0.014, n=3). The entrapment efficiency was determined by spectrofluorometer measurements. The efficiency was 44.4+/-1.6% for ICG and 74.3+/-1.9% for DOX. Drug loading was 0.015+/-0.001%, w/w, for ICG and 0.022+/-0.001%, w/w, for DOX (n=3). The release pattern was biphasic. ICG and DOX loaded-nanoparticle preparation was standardized based on the following parameters: PLGA concentration, PVA concentration and initial drug content.