Entrapment efficiency (EE) is a crucial parameter for the evaluation of nanocarriers. The accurate measurement of EE demands clear separation of nanocarriers from free drugs, which so far has not been clearly validated due to a lack of functional tools to identify nanocarriers. Herein, an environment-responsive water-quenching fluorophore was employed to label and identify model nanocarriers, polycaprolactone nanoparticles (PN), methoxy polyethylene glycol-poly(d,l-lactic acid) polymeric micelles (PM) and solid lipid nanoparticles (SLN). The separation process of three commonly used methods (centrifugation, ultrafiltration and gel permeation chromatography) was visualized by live imaging. The separation efficiency of the centrifugation method is very poor, especially for PM (40 nm), SLN (100 nm) and PN (100 nm); only PN (200 nm) can be efficiently separated but at a consumption of enormous energy. The ultrafiltration method shows the best separation efficiency with only 0.32–0.93% of leakage of the nanocarriers. Gel permeation chromatography exhibits good separation as well but suffers from low recovery, a potential factor that might compromise the accuracy of EE measurement. In conclusion, the ultrafiltration method is the method of choice for efficient separation and accurate measurement of EE.