In vitro activity measurements indicate that storage sweetpotato roots contain high amounts of extractable amylolytic enzymes. These storage roots also have a very high starch content, a characteristic indicating that the in vitro measurements estimate potential amylolytic activity rather than actual physiological activity. We are interested in optimizing the use of endogenous amylases when processing sweetpotato roots and have undertaken a study to identify physiological parameters that control in vivo starch breakdown. Sweetpotato roots were allowed to germinate for 35 days in controlled conditions. Using a combination of in vitro activity measurements and immunochemical detection, the spatial distribution and changes in activity levels for the three major amylolytic enzymes in storage sweetpotato roots—α-amylase, β-amylase, and starch phosphorylase—have been followed. After 6 days, α-amylase protein increased in the outer starchy parenchymatous tissues surrounding the cambium layers, a result suggesting a de novo synthesis of the enzyme in cambium or laticifers layers. β-Amylase was abundant throughout the root at all times, and its high levels did not directly affect starch degradation rates. Starch phosphorylase protein level remained constant, while its extractable activity increased. Starch content decreased during sweetpotato seed root germination. However, the amount of starch that disappeared during germination was low compared with the calculated starch hydrolysis potential estimated by amylolytic activity measurements.