Adverse outcome pathways (AOPs) are conceptual frameworks that organize and link contaminant-induced molecular changes to adverse biological responses at the individual and population level. The development of AOPs is a priority area of research in ecotoxicology as a means to leverage molecular and high content mechanistic information obtained through top-down ‘omic approaches in regulatory decision-making. Current AOPs for hormonally active agents (HAAs) focus on nuclear receptor-mediated effects despite the overwhelming evidence that HAAs also activate membrane receptors. Activation of membrane receptors triggers propagation of non-genomic signaling cascades often transduced by protein phosphorylation leading to phenotypic changes. To promote investigations of rapid non-genomic signaling mechanisms of environmental contaminants in aquatic species for top-down AOP development, we developed a phosphoproteomic pipeline using label-free LC-MS/MS. We used our optimized pipeline to identify proteins differentially phosphorylated in the brain of fathead minnows (Pimephales promelas) aqueously exposed for 30 minutes to two HAAs, 17α-ethinylestradiol (EE2), a strong estrogenic substance, and levonorgestrel (LNG), a progestin, both components of the birth control pill. Results revealed differential phosphorylation of proteins involved in neuronal processes such as nervous system development, synaptic transmission, and neuroprotection by EE2 while LNG induced differential phosphorylation of proteins involved in axon cargo transport and calcium ion homeostasis. EE2 and LNG caused similar enrichment of cell processes including synaptic plasticity and neurogenesis. This study is the first to identify molecular changes in vivo in fish after short-term exposure and highlights membrane receptors and transduction of rapid, non-genomic signaling mechanisms as targets of HAAs in addition to the well-established nuclear receptor-mediated pathways.