The development of plant root systems is characterized by a high plasticity, made possible by the continual propagation of new meristems. Root architecture is fundamental for overall plant growth, abiotic stress resistance, nutrient uptake, and response to environmental changes. Understanding the function of genes and proteins that control root architecture and stress resistance will contribute to the development of more sustainable systems of intensified crop production. To meet these challenges, proteomics provide the genome-wide scale characterization of protein expression pattern, subcellular localization, post-translational modifications, activity regulation, and molecular interactions. In this review, we describe a variety of proteomic strategies that have been applied to study the proteome of the whole organ and of specific cell types during root development. Each has advantages and limitations, but collectively they are providing important insights into the mechanisms by which auxin structures and patterns the root system and into the interplay between signaling networks, auxin transport and growth. The acquisition of proteomic, transcriptomic, and metabolomic data sets of the root apex on the cell scale has revealed the high spatial complexity of regulatory networks and fosters the use of new powerful proteomic tools for a full understanding of the control of root developmental processes and environmental responses.