Aim: This study examines the absolute quantification of particle uptake into cells. Methods: We developed a novel method to analyze stacks of confocal fluorescence images of single cells interacting with nano-and micro-particles. Particle_in_Cell-3D is a freely available ImageJ macro. During the image analysis routine, single cells are reconstructed in 3D and split into two volumes – intracellular and the membrane region. Next, particles are localized and color-coded accordingly. The mean intensity of single particles, measured in calibration experiments, is used to determine the absolute number of particles. Results: Particle_in_Cell-3D was successfully applied to measure the uptake of 80-nm mesoporous silica nanoparticles into HeLa cells. Furthermore, it was used to quantify the absolute number of 100-nm polystyrene nanoparticles forming agglomerates of up to five particles; the accuracy of these results was confirmed by super-resolution, stimulated emission depletion microscopy. Conclusion: Particle_in_Cell-3D is a fast and accurate method that allows the quantification of particle uptake into cells.