In comparison to the exponential increase of genotyping methods, phenotyping strategies are lagging behind in agricultural sciences. Genetic improvement depends upon the abundance of quantitative phenotypic data and the statistical partitioning of variance into environmental, genetic, and random effects. A metabolic phenotyping strategy was adapted to increase sample throughput while saving reagents, reducing cost, and simplifying data analysis. The chemical profiles of stem extracts from maize plants grown under low nitrogen (LN) or control trial (CT) were analyzed using optimized protocols for direct-injection electrospray ionization mass spectrometry (DIESI-MS). Specific ions significantly decreased or increased because of environmental (LN versus CT) or genotypic effects. Biochemical profiling with DIESI-MS had a superior cost-benefit compared to other standard analytical technologies (e.g., ultraviolet, near-infrared reflectance spectroscopy, high-performance liquid chromatography, and gas chromatography with flame ionization detection) routinely used for plant breeding. The method can be successfully applied in maize, strawberry, coffee, and other crop species.