Significance Nearly all proteins are posttranslationally modified, a phenomenon known to alter protein function. Recently, multiple posttranslational modifications (PTMs) have been documented to exist on the same proteins, revealing an additional level of complexity (named “PTM crosstalk”) that, due to its dynamic nature, is challenging to predict. Here, we propose a motif for PTM crosstalk between two of the most common PTMs: phosphorylation and O-GlcNAcylation. Through the use of a kinetic-based high-resolution mass spectrometry assay, we highlight specific residues that, when phosphorylated, hamper O-GlcNAcylation at nearby sites. In addition, we show that the Ser/Thr residues in one of the most common kinase motifs, PX(S/T)P, cannot be O-GlcNAcylated, demonstrating that reciprocal PTM crosstalk does not occur with Pro-directed kinases.