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We describe a label‐free proteomics protocol for the interrogation of the placental proteome. Step‐by‐step directions, including tissue cleanup and preparation, proteolytic digestion, nanoLC–MS/MS data collection and data analysis, are provided. The workflow has been applied toward exploring differential protein expression patterns in placentas from women who have been exposed to drugs during pregnancy relative to those who have not. We collected 20 tissue specimens, each representing a combination of spatially diverse sections across the placenta. These specimens were analyzed in the work described here, to survey information across the entire organ. This protocol can be scaled up or down as needed. [ABSTRACT FROM AUTHOR]