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ABA regulates stomatal movement by affecting ion transport in guard cells; yet in situ measurement of ABA-mediated dynamics of guard cell ion transport and the involvement of other phytohormones in regulating stomatal aperture under drought stress are still lacking. In this study, hydroponically grown plants of wild type barley Steptoe (WT) and its correspondent ABA-deficient barley mutant Az34 were treated with 10% polyethylene glycol (PEG) 6000 for 0, 2, 4, and 24 h or 9 d to mimic short- and long-term drought stress. The K+, H+ and Ca2+ fluxes in the guard cell were monitored in situ by noninvasive micro-test technology. Upon 10% PEG treatment, leaf ABA concentration ([ABA]leaf) of both barley genotypes increased dramatically after 2 h and reached the highest level after the 24 h. Compared to the control, a significant increase in Ca2+ influx in both genotypes was observed after 2 h exposure to PEG, and reached the largest value after 4 h in WT. The increase of [ABA]leaf coincided with the increase of K+ efflux and Ca2+ influx and the decrease of stomatal conductance in WT under short-term drought stress, though the concentrations of IAA, GA3 and ZR in WT were all increased at 4 h. K+ efflux of guard cells was significantly greater in WT than in Az34 at 24 h after PEG treatment. The results elucidate the role of ABA in mediating ion transports in guard cells, hereby regulating the stomatal movement in barley exposed to drought stress.