By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process - NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design. STATEMENT OF SIGNIFICANCE: By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.