Small well-defined spherical gold nanoparticles were synthesized by a simple non-physical method based on a mixture of gold salt, tetraethylene oxide and water, free of any additional reducing chemical agent or physical method. The ratio of tetraethylene oxide to water was optimized to achieve a fast synthesis within 30 min. Transmission electron microscopy images showed well dispersed gold nanospheres with a size ranging from 10 to 15 nm. XPS was used to confirm the oxidation state of gold nanoparticles and the oxidation products from tetraethylene oxide after the reaction. This new protocol performed in sustainable and biocompatible conditions is complementary to the current methods used to synthesize gold nanospheres. In order to use these particles in biological samples, we correlated the atomic absorption with the colorimetric concentration of nanospheres in solution. After 24 h of incubation of cancer or neuronal cell lines with these nanoparticles, transmission electron microscopy images showed similar cellular uptake in both cell lines, especially in cytoplasmic vesicular structures.