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N-Acylethanolamines (NAE) are a class of essential signaling lipids that are involved in a variety of physiological processes, such as energy homeostasis, anti-inflammatory responses, and neurological functions. NAE lipids are functionally different yet structurally similar and often have low concentrations in biological systems. Therefore, the comprehensive analysis of NAE lipids in complex biological matrices is very challenging. In this service, we used an ion mobility–mass spectrometry (IM-MS) based four-dimensional (4D) untargeted technology for comprehensive analysis of NAE lipids.