The ability of plants to uptake amino acids has been confirmed by extensive research; however, the subsequent post-uptake metabolism of absorbed amino acids is poorly characterized. With the aim of elucidating the different effects of inorganic and organic nitrogen sources on primary metabolic pathways in spinach, a gas chromatography–mass spectrometry (GC–MS) based metabolic approach was used to identify differentially abundant compounds in spinach (Spinacia oleracea) leaves after 2days hydroponic-culture in one of three concentrations of glycine or nitrate. Principal component analysis (PCA) revealed distinct clusters for different treatments. The first and second components of PCA accounted for 63.6% and 13.6% of total variance, and seemed to be mainly influenced by the nitrogen concentration and amount of Gly input, respectively. Compared to control treatment, most identified metabolites increased in dose-dependent manners as nitrogen availability increased; however, distinct patterns were observed for NO3−- and Gly-treated leaves. When nitrogen was deficient, NO3−-treated leaves had lower relative contents of carbohydrates, organic acids and amino acids than Gly treated leaves, this trend reversed under N-sufficient conditions. Furthermore, the metabolites identified were profiled to highlight the overall metabolic shifts between NO3− and Gly as N sources at different concentrations.