Amiodarone is a drug that is widely used in the treatment of heart disease. To circumvent side effects, colloidal drug carriers have been designed to deliver the drug specifically to the site of action. For the purposes of in vitro characterization of such particles, difficult test systems are employed that usually require the quantitative separation of the drug carrier from the release medium before analysis. In this work, a Langmuir balance was used to characterize amiodarone release. Drug-loaded nanoparticles were prepared from a biodegradable polyester and assayed for their drug release kinetics. Simultaneously, nanoparticles were analyzed for their drug release by a standard procedure based on dialysis tubes combined with high-performance, liquid chromatography. The results obtained by the Langmuir balance experiments were compared with those obtained from high-performance liquid chromatography and were found to correlate well. The interexperimental variation was 4.4% for the Langmuir method (n = 4), and the interexperimental variation for HPLC was 2.9% (n = 3). The major advantage of this new method is the possibility diminishing significantly the required sample amount for the experiment, allowing drug detection in the lower nanomolar range. Moreover, the avoidance of prior nanoparticle separation from the release medium provides important progress of this technique. The Langmuir balance has proven its adaptability as a new sensitive tool for the characterization of amphiphilic drug release kinetics.