Given the output MD trajectories, all metrics were measured for each glycoform structure. They were later compared with the experimental work for assessing the efficacy. Notably, we found that at minimum 5 × 2000 ns = 10 𝜇s of simulation was necessary to capture the structural characteristics of the insulin ensembles. This aggregate length is, to our knowledge, one of the longest among all the computational studies of human insulin. In addition, our study is not only one of the very few studies that characterize conformations of insulin with a covalently attached moiety, but is also unique as the first study that correlates molecular dynamics simulations to the proteolytic stability of these proteins. Our investigation facilitates a better understanding of the underlying mechanism of how different O-glycosylation patterns influence insulin properties, which could be useful in guiding the design of insulin glyco-variants with better properties for oral delivery.