This dataset contains TMT-based quantitative phosphoproteomics data generated from neonatal mouse ventricular myocytes (NMVMs) comparing control cells versus NUAK1 overexpression (three biological replicates per group; CTL_1–3 and OE_1–3). Samples were analyzed by LC–MS/MS with fractionated runs (raw fractions F1–F3). MS/MS spectra were searched using MaxQuant (v1.6.15.0) against the UniProt Mus musculus reference proteome (release 2024-06-04). Enzyme was set to Trypsin/P. Fixed modification: Carbamidomethyl (C). Variable modifications: Oxidation (M), Protein N-term acetylation, and Phospho (STY). Mass tolerances were set to 20 ppm (first search) and 5 ppm (main search) for precursor ions and 20 ppm for fragment ions. Protein and PSM FDR were controlled at 1%. The submission includes the raw files and identification/quantification summary tables used for downstream QC and differential phosphorylation analyses.