Early lactation is a very critical phase in the nutrition and management of dairy cows. To alleviate any negative energy balance during early lactation cows are fed high proportion of cereal grains. Transitioning from a diet of grass and hay to one consisting primarily of grains cause the incidence of many metabolic disorders (ketosis, fatty liver and rumen acidosis). However, the precise mechanism(s) of how grain feeding alters the rumen environment and causes metabolic disorders is not clear. Because metabolomics is such a powerful approach for studying the chemical changes in biological systems, we undertook a comprehensive, quantitative metabolomics analysis of rumen fluid samples taken from 8 dairy cows fed graded amount of barley grain (i.e., 0, 15, 30, and 45% of diet dry matter).By using improved metabolite detection techniques, including proton NMR, GC-MS, and DFI-MS, we identified and quantified 93 metabolites in 4 different ruminal fluid samples. Data collected by these advanced metabolomics techniques, was analyzed by MetaboAnalyst (an online tool for metabolomic data analysis).Multivariate analysis showed that when cows fed with high-grain diets (>30%) resulted in increased rumen fluid concentrations of several toxic, inflammatory, and unnatural compounds including putrescine, methylamines, ethanol, glucose, urea, ethanolamine, and short-chain fatty acids. Perturbations in several amino acids (phenylalanine, ornithine, lysine, leucine, arginine, valine, and phenylacetylglycine) were also observed. By using ANOVA, it was also revealed a drop in ruminal pH and a decreased concentration of 3-phenylpropionate in cows fed greater amounts of cereal grain. These results certainly underline the importance of gaining a better understanding of the biochemical function of rumen as a whole ecosystem. Deeper understanding of how diet influences rumen health, as well as improved methods for monitoring these changes should enable us to maintain the fine balance between high milk productivity and good herd health.