Microglia are the main immune cells in the brain playing a critical role in neuroinflammation, and numerous pieces of evidence have proved that energy metabolism is closely associated with inflammation in activated microglia. Salidroside (Sal) isolated from Tibetan medicine Rhodiola crenulate can inhibit microglial hypoxia inflammation (HI). However, whether the inhibition is due to the intervening energy metabolic process in microglia is not clear. In this work, the hypoxic microenvironment of BV2 microglial cells was simulated using deferoxamine (DFO) in vitro and the change of cell metabolites (lactate, succinate, malate, and fumarate) was real-time online investigated based on a cell microfluidic chip-mass spectrometry (CM-MS) system. Meanwhile, for confirming the metabolic mechanism of BV2 cells under hypoxia, the level of HI-related factors (LDH, ROS, HIF-1α, NF-κB p65, TNF-α, IL-1β, and IL-6) was detected by molecular biotechnology. Integration of the detected results revealed that DFO-induced BV2 cell HI was associated with the process of energy metabolism, in which cell energy metabolism changed from oxidative phosphorylation to glycolysis. Furthermore, administration of Sal treatment could effectively invert this change, and two metabolites of Sal were identified: tyrosol and 4-hydroxyphenylacetic acid. In general, we illustrated a new mechanism of Sal for reducing BV2 cell HI injury and presented a novel analysis strategy that opened a way for real-time online monitoring of the energy metabolic mechanism of the effect of drugs on cells and further provided a superior strategy to screen natural drug candidates for HI-related brain disease treatment.