Chitosan nanoparticles prepared by ionotropic gelation with the tripolyphosphate (TPP) polyanion have been widely considered for drug delivery. The stability (shelf-life) of TPP-chitosan nanoparticles is highly relevant to its potential use as a drug delivery agent as this plays an important role in the function of the nanoparticle and will determine shelf-life. In the present study, the physical stability (in terms of particle size) of TPP-chitosan nanoparticles was measured across a range of different temperature conditions: 4 °C, 25 °C and 40 °C using differential sedimentation. After 12 months storage at 4 and 25 °C the size of nanoparticles remained similar to those of the freshly prepared samples, whilst after storage at 40 °C there were little or no TPP-chitosan nanoparticles remaining after only 6 months. This may be due to the decrease in molar mass of the chitosan possibly due to hydrolysis causing scission of the polymer chains, which results in a decrease in nanoparticle size and eventual disintegration. This mechanism is important in the application of TPP-chitosan as a drug delivery agent.