Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its biocompatibility, high charge density, nontoxicity, and mucoadhesion properties. Processing techniques for the preparation of chitosan microspheres have been extensively developed since the 1980s. The present paper describes for the first time a fast and one-step process for the preparation of stable chitosan microspheres by a simple sonochemical method. The microspheres were characterized by their particle size, surface morphology, stability, and drug-entrapment efficiency. The average size of the microspheres was found to be around 1 mum with a narrow size distribution, which enabled them to be used for in vivo applications. The encapsulation of different dyes into these microspheres was readily achieved with more than 75 % efficacy by dissolving them into the organic phase before sonication. The chitosan microspheres demonstrated excellent stability toward acidic and basic conditions ranging from pH 4 to 9, thereby indicating their implementation as possible therapeutic and diagnostic agents. The stability of these microspheres appears to be contributed from intermolecular imine cross-linking in addition to other noncovalent interactions. The ability of the surface-exposed amino groups of chitosan microspheres to undergo chemical conjugation with potential drugs and/or targeting vectors was determined by their reaction with fluorescein isothiocyanate (FITC) and fluorescamine followed by confocal microscopy.