Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to improve the monoclonal antibody-based therapy, attempts have been focused in the formulation of these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography with fluorescence detection method for the determination of bevacizumab from nanoparticulate systems, according to the International Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP-C8 column with a mobile phase composed by water-0.1% (v/v) TFA and acetonitrile-0.1% (v/v) TFA in gradient mode at a flow rate of 1 mL min−1. Results showed that the proposed method is specific, linear in the range of 10–100 μg mL−1 (r2 = 0.9997), accurate (recovery rate 100.50 ± 0.85%), precise at the intraday and inter-day (relative standard deviation less than 1.79%) and robust. The detection and quantification limits were calculated by specific linear calibration curve with less concentrated standard (range of 1–20 μg mL−1). The LOD was 2.16 μg mL−1 and LOQ was 6.55 μg mL−1. This method was also successfully used, for the first time, to quantify and compare the content of bevacizumab encapsulated into poly(lactic-co-glycolic acid)-based nanoparticles before and after lyophilization.