Biodiesel is a renewable source of global interest and one of the most attractive alternatives to partially replace petroleum fuels. Because of its chemical nature, however, biodiesel quality may be more rapidly degraded (than hydrocarbon-based fossil fuels) because of oxidation particularly during long or inappropriate storage. Herein, we show that a direct, rapid, and easy to implement and perform technique, that is, easy ambient sonic-spray ionization mass spectrometry (EASI-MS), can be successfully applied to monitor biodiesel oxidation. Soybean biodiesel stored under different conditions has been monitored by EASI-MS, and markers of oxidation have been identified. A new oxidation index (I-ox) rapidly and easily determined via EASI-MS was proposed.