Due to the limited available amounts of components, especially of low water-soluble drugs, formulation development is often impeded by a careful characterization. The use of small batch sizes might solve this problem but requires also adequate analytics. Concentration of nanoparticulate formulations lack straightforward evaluation techniques. In this work, a precise and straight-forward method is established to individually count nanoparticles. A microfluidic chip with known dimensions was used to visualize single particles flowing through the channel (single-particle tracking (SPT)). A sequence of 10,000 images was analyzed to determine the mean particle concentration. The proposed method is independent of the particular flow rate through the microfluidic chip as long as there is no particle overlap and a continuous exchange of particles. Monodisperse Rhodamine B labeled poly (methyl methacrylate) (PMMA) nanoparticles (267.03 ± 9.79 nm) were used as a model and reference particle system for the evaluation process of SPT allowing for a gravimetric determination based on density analysis using analytical ultracentrifugation (AUC) and gas pycnometry. The SPT method was evaluated and compared to other techniques used for concentration measurement. Both approaches (SPT and gravimetry) provide very similar and comparable results indicating the applicability of this novel quantification approach. In contrast, multi angle dynamic light scattering (MADLS) could not yield a precision as good as SPT (number density rel. standard deviation SD nSPT = 11.67%; SD nMADLS = 49.45%). Finally, the measured particle number concentrations can be realized in low concentration ranges (0.8249 μg mL−1 – 0.08249 μg mL−1) not accessible for MADLS (0.08249 mg mL−1 – 0.008249 mg mL−1) and gravimetric analysis.