Due to the considerable amount of applications of gold nanoparticles (AuNPs) in biological systems, there is a great need for an improved methodology to quantitatively measure the uptake of AuNPs in cells. Flow cytometry has the ability to measure intracellular AuNPs by collecting the light scattering from a large population of live cells through efficient single cell analysis. Traditionally, the side scattering setting of the flow cytometer, which is associated with a 488 nm excitation laser (SSC channel), is used to detect nanoparticle uptake. This method is limited as AuNPs do not have the optimized response when excited with this laser. Here, we reported that the use of more red-shifted excitation lasers will greatly enhance the optical signal needed for the flow cytometry-based detection of AuNSs (26 nm in diameter) and AuNRs (67 nm × 33 nm, length × width) uptake in triple negative breast cancer cells (MDA-MB-231).