This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of β-actins at the single-cell level were quantified as 14.2 ± 1.7 μm and 9.62 ± 4.29 × 105 (A549, ncell = 14 242), 13.0 ± 2.0 μm and 6.46 ± 3.34 × 105 (Hep G2, ncell = 35 932), 13.8 ± 1.9 μm and 1.58 ± 0.90 × 106 (MCF 10 A, ncell = 16 650), and 12.7 ± 1.5 μm and 1.09 ± 0.49 × 106 (HeLa, ncell = 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.