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Adolescence is hypothesized to be a critical period for the development of association cortex. A reduction of the excitation:inhibition (E:I) ratio is a hallmark of critical period development; however, it has been unclear how to assess the development of the E:I ratio using noninvasive neuroimaging techniques. Here, we used pharmacological fMRI with a GABAergic benzodiazepine challenge to empirically generate a model of E:I ratio based on multivariate patterns of functional connectivity. In an independent sample of 879 youth (ages 8 to 22 years), this model predicted reductions in the E:I ratio during adolescence, which were specific to association cortex and related to psychopathology. These findings support hypothesized shifts in E:I balance of association cortices during a neurodevelopmental critical period in adolescence.