Hydroponic cultivation is a soil-free technique commonly used in agriculture where plants are grown in a highly controlled environment leading to high crop yields. Hydroponic Isotope Labeling of Entire Plants (HILEP) is the most cost-effective isotope labeling method for quantitative plant proteomics, enabling the metabolic labeling of whole and mature plants with a stable isotope such as 15N. Employing inorganic 15N-containing salts as the sole nitrogen source, healthy plants can be easily grown and labeled in hydroponic solutions. Close to 100% 15N-labeling of proteins can be achieved using HILEP. Moreover, hydroponic cultivation allows tight control of growth conditions. Plants grown in 14N- and 15N-hydroponic media are typically pooled straight after harvest, eliminating any bias due to subsequent sample preparation and analysis. The pooled 14N-/15N-protein extracts can be fractionated in any convenient way and digested with trypsin (or any other enzyme of choice). Peptides can then be analyzed by techniques such as liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Following protein identification, the spectra of 14N/15N-peptide pairs are typically compared and relative protein amounts are calculated from the 14N/15N-ion signal ratios. An increasing number of bioinformatics tools are now available for determining these ratios in a convenient way.