Casein kinase 1 (CK1) is a highly conserved and ubiquitous serine/threonine protein kinase in eukaryotic cells. AELs (Arabidopsis EL1-like) are plant-specific CK1s and function in a wide range of physiological and signaling processes. To comprehensively understand the cellular functions and regulatory mechanism of AELs-mediated phosphorylation, we conducted a quantitative data-independent acquisition (DIA) based phosphoproteomic assay. A total of 3107 phosphopeptides and 1032 putative substrates were detected and enriched novel phosphorylation motifs were identified, which greatly expanded the candidate substrates and functions of CK1, as well as those from human and rice.