The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI(-)) and NG(+)/TMRE(+) when compared to NB1. Stimulation Indices (SI) for Liberase HI (n = 45) showed 3.17 and NB1 (n = 18) 2.71 (p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable β cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.