Plant health and development are directly depending on a plant's ability to react to a constantly changing environment. Sensing of water and nutrition levels and of the biotic environment is vital for a plant, making the root one of the key plant organs. Proteins are the key molecules that play numerous roles in a cell's everyday life. Quantitative proteome profiling of roots can provide a global overview on the molecular regulatory mechanisms and networks involved in plant growth and development and abiotic and biotic stress responses. Here, we provide a detailed proteomics workflow on Arabidopsis thaliana roots from plant growth up to proteomics data analysis.