Increased plant tolerance to stress may be chemically induced with applications of salicylic acid (SA). The aim of this study was to determine the change in the SA leaf concentration over time in response to the SA spraying in leaves of greenhouse grown tomato. In sprayed leaves the SA concentration showed changes over time similar to the reported responses to environmental stress. Two days after the first application, the SA foliar concentration reached the maximum of 8 µg•g −1 , equivalent to twice the amount observed in the control plants. SA decreased until it reached the level of control plants eight days later. A second application showed actually the same response, but with a faster decline of SA in two days. According to the results of this assay, SA applications on tomato should be performed within a minimum interval of eight days in order to maintain the SA concentration related with the increase in plant tolerance to environmental stress.