Polymer masked-UnMasked Protein Therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid or poly(l-glutamic acid), to mask a protein or peptide’s activity; subsequent locally-triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. While the concept of PUMPT is well-established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography (SEC) was employed to collect fractions of differentially degraded conjugate and ultra performance liquid chromatography mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy (CLSM) of LIVE/DEAD® stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an MTT assay. UPLC-MS revealed that fully ‘unmasked’ dextrin-colistin conjugate comprised of colistin bound to at least one linker, while larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully ‘unmasked’ conjugate compared to the partially-degraded species (MIC = 0.25 and 2-8 µg/mL, respectively), while dextrin conjugation reduced colistin’s in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.