The major stress-inducible Hsp70 (HSPA1A) is overexpressed in highly aggressive tumor cells including glioblastoma multiforme and presented on their plasma membrane. Ionizing irradiation increases cytosolic and membrane-bound Hsp70 (mHsp70) on tumor cells. Depending on its intracellular or membrane localization, Hsp70 either promotes tumor growth or serves as a target for the innate immune system. To define the optimal window for a therapy with Hsp70-tageting NK cells the kinetics of the mHsp70 density on the plasma membrane of human glioma cells (U87) has been studied after radiotherapy by multiparameter flow cytometry. Cell cycle dependent alterations in the mHsp70 expression were excluded by seeding different cell numbers on day 0. Low dose irradiation (2 Gy) results in a slow upregulation of the mHsp70 density in U87 cells which peaks on day 4 and starts to decline from day 7 onwards. In contrast, higher radiation doses (4 Gy, 6 Gy) resulted in a faster upregulation of mHsp70 expression on days 2 and 1, respectively, followed by a decline from day 5 onwards. Functionally, elevated mHsp70 densities correlate with an improved lysis by Hsp70-targeting NK cells. In summary, the kinetics of changes in the mHsp70 density upon irradiation on tumor cells is time and dose-dependent.