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Non-specific disruption of cellular membranes induced by aggregation of exogeneous β-amyloid (Aβ) peptides is considered a viable pathological mechanism in Alzheimer's disease (AD). The solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied in model liposomes to provide important insights on the molecular interactions between membranes and Aβ aggregates. Yet, the feasibility of in-cell ssNMR spectroscopy to probe Aβ-membrane interactions in native cellular environments has rarely been tested. Here we report the application of in-cell 31P ssNMR spectroscopy on live mouse neuroblastoma Neuro-2a (N2a) cells under moderate magic angle spinning (MAS) conditions. Both cell viability and cytoplasmic membrane integrity are retained for up to six hours under 5 kHz MAS frequency at 277 K, which allow applications of direct-polarization 31P spectroscopy and 31P spin-spin (T 2) relaxation measurements. The 31P T 2 relaxation time constant of N2a cells is significantly increased compared with the model liposome prepared with comparable major phospholipid compositions. With the addition of 5 μM 40-residue Aβ (Aβ 1 – 40) peptides, the 31P T 2 relaxation is instantly accelerated. This work demonstrates the feasibility of using in-cell 31P ssNMR to investigate the Aβ-membrane interactions in the biologically relevant cellular system. [Display omitted] • Neuro-2a cells maintain viability and integrity for six hours under moderate magic angle spinning conditions. • 31P NMR spectroscopy revealed partial resolution for the glycerol- and lyso-phospholipid resonance peaks. • 31P T 2 relaxation is significantly slower in N2a cell samples compared with model phospholipid vesicles. • 31P T 2 relaxation is slightly more rapid with the exogeneous β-amyloid peptides. [ABSTRACT FROM AUTHOR]