The aim of this study was to prepare docetaxel-loaded chitosan microspheres and to evaluate their in vitro and in vivo characteristics. Glutaraldehyde crosslinked microspheres were prepared using a water-in-oil emulsification method, and characterized in terms of the morphological examination, particle size distribution, encapsulation ratio, drug-loading coefficient and in vitro release. Pharmacokinetics and biodistribution studies were used to evaluate that microspheres have more advantage than the conventional formulations. The emulsion crosslinking method was simple to prepare microspheres and easy to scale up. The formed microspheres were spherical in shape, with a smooth surface and the size was uniform (9.6 +/- 0.8 m); the encapsulation efficiency and drug loading of prepared microspheres were 88.1% +/- 3.5% and 18.7% +/- 1.2%, respectively. In vitro release indicated that the DTX microspheres had a well-sustained release efficacy and in vivo studies showed that the microspheres were found to release the drug to a maximum extent in the target tissue (lung). The prepared microspheres were found to possess suitable physico-chemical properties and the particle size range. The sustained release of DTX from microspheres revealed its applicability as drug delivery system to minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in target sites.