Aberrant phosphorylation and aggregation of tau protein are among the core pathological features of Alzheimer's disease (AD). The P301L mutation, located in the microtubule-binding repeat region of tau protein, significantly reduces the affinity of tau for microtubules, thereby inducing hyperphosphorylation, aggregation into paired helical filaments and neurofibrillary tangles, ultimately accelerating neuronal degeneration. To more realistically simulate the pathological microenvironment of AD, this study selected human hippocampal neurons as the expression host. This brain region highly corresponds to the most clinically affected areas of AD, thus more accurately reflecting the molecular responses of human neurons under pathological conditions. Based on this cell model, this study used affinity peptide magnetic beads to enrich N-phosphorylated peptides before and after transfection under neutral conditions, and systematically identified them using high-resolution liquid chromatography-tandem mass spectrometry (HR-LC-MS/MS), identifying a total of 6064 N-phosphorylation sites. By comparing the differences in site modifications before and after the P301L mutation, this study systematically elucidated the molecular mechanism by which this mutation drives early pathological events such as tau protein aggregation, synaptic dysfunction, and neural network overexcitation through differential modification of specific phosphorylation sites, providing a rich data foundation for related research. Furthermore, by combining quantitative proteomics techniques, this study tracked the dynamic evolution of N-phosphorylation modifications during the course of Alzheimer's disease (AD), revealing its potential regulatory patterns in disease progression and providing new experimental evidence and theoretical perspectives for a deeper understanding of AD pathogenesis. In summary, this study not only expands our understanding of the functional role of N-phosphorylation modifications in the pathological process of AD but also provides key candidate targets and detailed reference data for the screening of early diagnostic biomarkers for AD and the development of drugs targeting tau phosphorylation, possessing both significant basic research value and clinical translational potential.