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Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.