Absolute quantitative measurements of nanoparticle (NP) uptake are a prerequisite to determine doses of NPs in pharmacological and toxicological studies. However, absolute quantitation is rarely reported, hindering the comparison between different studies. Here, a new flow cytometric approach is presented to analyze fluorescent NPs with a “standard” non-scanning flow cytometer and to quantify them inside cells. The mean fluorescence intensity of a single particle and the particle concentration (NPs per μL medium) are obtained. A routine for rapid quantitative counting of the endocytosed NPs in HeLa cells by flow cytometry (FC) is developed and validated by confocal laser scanning microscopy. As a proof-of-concept, the quantitative measurements show that the cellular uptake efficiency of negatively charged poly(methyl methacrylate) NPs is very low, that is, in the range of 10−3% of the added particle amount.