<p>For the development of cancer diagnostics and monitoring of cellular metabolism, sensitive and label-free analytical tools are required. In this work, we report the development of silver nanostructures through femtosecond (fs) laser processing, which allows for the detection of metabolic changes in cancer cells with the aid of surface-enhanced Raman scattering (SERS) efficiently. SEM, AFM, and UV-Vis spectroscopy all confirmed that the ultrafast laser technique formed unique, reproducible plasmonic nanostructures with strong electromagnetic hotspots. These substrates showed excellent consistency and enhancement factors, providing a reliable platform for biomolecular fingerprint probing. When Ag nanostructures formed were employed for the real-time metabolic monitoring of cancer and normal cells, various Raman signals corresponding to proteins, lipids, and nucleic acids were observed. Principal component analysis (PCA) and hierarchical clustering analysis (HCA), being two multivariate statistical analyses, were capable of discriminating between cancer and normal cells, proving the sensitivity of the method to subtle biochemical alterations. The SERS response was also found to be very stable and reproducible, suggesting that it can be utilized for dynamic and quantitative cellular investigations. All in all, our observations legitimize Ag nanostructures fabricated by fs lasers as a versatile and non-invasive SERS platform for research on cancer cell metabolism. Synergizing this technique with microfluidics and machine learning may result in high-throughput, automated, and clinically valuable cancer diagnosis. This study provides the foundation for the development of next-generation precision diagnostic tools in biomedical plasmonics and proves the plasmonic activity of ultrafast laser-created nanostructures.</p> Graphical Abstract <p>The graphical abstract illustrates the overall workflow of the study. Femtosecond (fs) laser pulses are used to fabricate silver (Ag) nanostructures, which act as plasmonic substrates. These nanostructures enable plasmon-enhanced surface-enhanced Raman scattering (SERS) for highly sensitive detection of biomolecular signatures. Cancer cells interacting with the Ag nanostructures produce distinct Raman signals that reflect metabolic dynamics. Advanced chemometric analyses, including PCA and HCA, are applied to distinguish spectral variations and classify cell states. The schematic highlights the integration of ultrafast laser nanofabrication with real-time SERS for monitoring cancer cell metabolism.</p> <p></p>