This letter reports on the investigation of the mechano-chemical effect of cross-linked dried free-standing alpha-lactalbumin (α-lactalbumin) thin films induced by different cation, calcium, magnesium, and potassium binding. The protein membranes were fabricated by drying droplets of an α-lactalbumin solution on top of silicon through-wafer holes obtained by deep reactive ion etching. Then the membranes were consecutively exposed to solutions of the cations in HEPES buffer solution while their resonant frequencies were measured by full-field surface stroboscopic white light interferometry. Tests on more than 30 free-standing protein films showed more significant conformational changes of the α-lactalbumin after immersion in a calcium solution than those observed after immersion in magnesium and potassium solutions. These results demonstrate, the real potential of free-standing protein films to be used as resonant biosensors for multiple cation detection.