Glucosinolates are nitrogen- and sulfur-containing specialized metabolites found in the plant order Capparales, which includes cruciferous plants. Glucosinolates and their degradation products play important roles in plant defense and in interactions with the environment. Here we report the effect of soil water stress on glucosinolate content in rosette leaves of Arabidopsis thaliana during the vegetative stage. The results showed that the water stress treatment caused a significant reduction in glucosinolate levels in rosette leaves of A. thaliana compared to well-irrigated control samples after 3 days of treatment. The differences between control and treatment became increasingly more significant over time, with both total and aliphatic glucosinolate content affected. Indole glucosinolates did not exhibit significant response to water stress. The content of 4-methylsulphinylbutyl glucosinolate (4MSOB) in rosette leaves was the highest among all the aliphatic glucosinolates detected. The decrease in the contents of 4MSOB after water stress is a main factor that affected the overall glucosinolate profile in the rosette leaves.