Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol allows also separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods ranging from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shotgun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared to the utilization of commercial kits, and its performance over a wide set of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method to a universal alternative for multiple molecular isolation from plant tissues.This article is protected by copyright. All rights reserved.