To scrutinize materials for specific biomedical applications, we need sensitive and selective analytical methods that can give more insight into the process of their biodegradation. In the present study, the enzymatic degradation of multi-block poly(ester amide) based on natural amino acids, such as lysine and leucine, was performed with serine proteases (alpha-chymotrypsin (alpha-CT) and proteinase K (PK)) in phosphate-buffered saline solution at 37 degrees C for 4 weeks. Fully and partially degraded water-soluble products were analyzed by liquid chromatography hyphenated with time-of-flight mass spectrometry using an electrospray interface (LC-ESI-ToF-MS). Tracking the release of monomeric and oligomeric products into the enzyme media during the course of enzymatic degradation revealed the preferences of alpha-CT and PK toward ester and amide bonds: both alpha-CT and PK showed esterase and amidase activity. Although within the experimental time frame up to 30 and 15% weight loss was observed in case of alpha-CT and PK, respectively, analysis by size exclusion chromatography showed no change in the characteristic molecular-weight averages of the remaining polymer. This suggests that the enzymatic degradation occurs at the surface of this biomaterial. A sustained and linear degradation over a period of 4 weeks supports the potential of this class of poly(ester amide)s for drug delivery applications.