This study reports a tailored workflow to detect and assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. The workflow involves a specific sample preparation and mass spectrometry approach to enrich and identify pyrophosphopeptides. A total of 148 pyrophosphosites across 71 human proteins were manually validated, with nucleolar proteins NOLC1 and TCOF1 being the most heavily pyrophosphorylated. The detection was consistent with biochemical evidence linking pyrophosphorylation to inositol pyrophosphates (PP-InsPs). Disruption of PP-InsP biosynthesis significantly reduced rDNA transcription, suggesting a role for pyrophosphorylation in regulating this process. The study highlights protein pyrophosphorylation as a significant non-canonical phosphorylation and emphasizes its importance in future phosphoproteomic analyses.This study reports a tailored workflow to detect and assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. The workflow involves a specific sample preparation and mass spectrometry approach to enrich and identify pyrophosphopeptides. A total of 148 pyrophosphosites across 71 human proteins were manually validated, with nucleolar proteins NOLC1 and TCOF1 being the most heavily pyrophosphorylated. The detection was consistent with biochemical evidence linking pyrophosphorylation to inositol pyrophosphates (PP-InsPs). Disruption of PP-InsP biosynthesis significantly reduced rDNA transcription, suggesting a role for pyrophosphorylation in regulating this process. The study highlights protein pyrophosphorylation as a significant non-canonical phosphorylation and emphasizes its importance in future phosphoproteomic analyses.