Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The properties of these nanomaterails towards in vitro biodegradation are very important and usually are studied using the model physiological conditions. In this work the novel approach based on application of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process) also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for degradation of polyester and polypetide-based particles was demonstrated and compared to the process observed in human blood plasma.