When synchronized with the light/dark cycle the circadian rhythm is termed a diurnal rhythm and this organizes an organism's daily life cycle in relation to the metabolic shifts during the day/night cycles. This is a complex task, particularly under stress conditions. Accurate maintenance of the diurnal rhythm becomes an issue under environmental extremes, such as drought due to the impairment of metabolism, redox balance, and structural integrity. In plants, the non-proteinogenic amino acid GABA accumulates to high levels in response to several stress factors but this is not always dependent on the activation of its biosynthesis. Here we propose a regulatory role to GABA during the diurnal rhythm in plants which is similar to its function in animals where it adjusts the circadian rhythm. Here we investigated whether GABA-biosynthesis was affected by drought stress during the diurnal cycle. For this, we took samples from leaves of N. tabacum plants subjected to PEG-mediated drought stress (-0.73 MPa) during the day and night cycle during a 24 hour period. Glutamate, GABA, and proline contents, along with GDH, GAD enzyme activities and transcript profiles were analyzed. Overall, we conclude that the oscillations in GABA biosynthesis during day and night cycle have an impact on drought stress responses which needs to be elucidated by further analysis.