The characteristics of Ca²⁺ and H⁺ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H⁺ concentration and increase in cytoplasmic Ca²⁺ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca²⁺ and H⁺ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca²⁺ and H⁺ fluxes across transformed Pichia pastoris cells in response to glycerol and polyethylene glycol 6000 (PEG6000) treatments. The transformed yeast cells are loaded onto a coverslide pre-processed in the poly-L-lysine solution (0.1% w/v aqueous solution). After cell immobilization, microelectrodes are positioned above a monolayer of attached cell population. Micro-volts differences are measured at two excursion points manipulated by a computer. Micro-volts differences could be converted into ion fluxes using the ASET 2.0 and iFluxes 1.0 Software. The method is expected to promote the application of NMT in microbiology. We are very grateful to Younger USA (Xuyue Beijing) NMT Service Center for their critical reading of the manuscript.