The biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) has been extensively utilized and investigated as a drug delivery system. Although in vivo biodegradation (at specific administration sites only) of PLGA-based drug delivery constructs, such as foams and microparticles, has been studied, quantitative in vivo biodegradation of distributed polymer nanoparticles has not been accomplished and is quintessential for designing formulations to achieve desired pharmacokinetic properties of a drug in a target tissue. We determined the in vivo degradation kinetics of PLGA nanoparticles, of two sizes, distributed in liver, spleen, and lungs following intravenous administration. In addition, we simultaneously determined the amount of polymer in tissues. Nanoparticle degradation in vitro and in vivo appears to be a first-order process, and useful correlations were obtained between in vitro and in vivo tissue degradation of the nanoparticles. The ability to detect in vivo degradation and biodistribution of polymer nanoparticles is a significant milestone for the rational design of degradable nanoparticle-based drug delivery systems capable of delivering the therapeutic agent in a closely predictable manner to target tissue.