This study investigates the uptake and retention of stable fluorescent organosilica nanoparticles by macrophages, which play a vital role in scavenging environmental nanoparticles and nanomedicines within the body. We used rhodamine 6G-loaded fluorescent organosilica nanoparticles (SiNP-R6G) synthesized from a thiol-functionalized organosilane precursor. Our primary objective was to establish a quantitative relationship between fluorescent measurements and nanoparticle tracking analysis, enabling the precise "counting" of nanoparticles taken up by macrophages under kinetic measurement conditions. Our kinetic study demonstrated a concentration-dependent, saturable internalization of nanoparticles in a model macrophage (RAW 264.7 cells), with a maximum uptake rate (V max ) of 7.9 × 10 4 nanoparticles per hour per cell. The estimated number concentration of nanoparticles for half-maximum uptake was approximately 0.8 trillion nanoparticles per milliliter, and a significant portion (∼80%) of internalized SiNP-R6G remained entrapped within the cells for 48 h, indicating the sustained particle retention capacity of macrophages. Our data further demonstrate that the internalized nanoparticles occupy more than 10% of the total cell volume, without inducing cytotoxic effects. This heightened capacity for nanoparticle uptake is attributed to intracellular vesicles capable of accommodating up to 50 densely packed, yet nonaggregated, nanoparticles within a single vesicle. In summary, our study underscores the effective development of a methodology for precise "counting" of cellular nanoparticle uptake in macrophages, providing valuable insights into the kinetics and retention capabilities of macrophages for nanoparticle-based drug delivery.