The implementation of non-destructive methods for the study of water changes within plant tissues and/or organs has been a target for some time in plant physiology. Recent advances in air-coupled ultrasonic spectroscopy have enabled ultrasonic waves to be applied to the on-line and real-time assessment of the water content of different materials. In this study, this technique has been applied as a non-destructive, non-invasive, non-contact, and repeatable method for the determination of water status in Populusxeuramericana and Prunus laurocerasus leaves. Frequency spectra of the transmittance of ultrasounds through plant leaves reveal the presence of at least one resonance. At this resonant frequency, transmittance is at its maximum. This work demonstrates that changes in leaf relative water content (RWC) and water potential (Psi) for both species can be accurately monitored by the corresponding changes in resonant frequency. The differential response found between both species may be due to the contrasting leaf structural features and the differences found in the parameters derived from the P-V curves. The turgor loss point has been precisely defined by this new technique, as it is derived from the lack of significant differences between the relative water content at the turgor loss point (RWC(TLP)) obtained from P-V curves and ultrasonic measurements. The measurement of the turgor gradient between two different points of a naturally transpiring leaf is easily carried out with the method introduced here. Therefore, such a procedure can be an accurate tool for the study of all processes where changes in leaf water status are involved.