Lily bulbs, with glycosides as the main components, have a long history of application as both food and medicine. This study investigated the protective effect of total glycosides extracted from lily (TGL) on mitochondrial damage in heart H9C2 cells induced by H2O2, and identified its ingredients using ultra-high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MSn) method. The results showed that TGL could reduce H9C2 cells viability damage and protect the cell morphology. Further investigations demonstrated that TGL could inhibit the decrease of mitochondrial membrane potential (Delta Psi M) and alleviate the decrease of ATP caused by H2O2. Total 31 compounds with potential protective effect of TGL were identified. These findings suggest that TGL has cardiomyocyte protective activity possibly by protecting the mitochondria. The established UHPLC-LTQ-Orbitrap-MSn method would be a dependable analytical tool for characterizing constituents in TGL and make the pharmacological experiments repeatable.