Quantitation of cytochrome c (Cyt c) in cell lysates through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs) as the matrix and GR-10 peptide as an internal standard has been demonstrated. To shorten digestion time, temperature sensitive microgels containing trypsin (TR) and Au NPs have been employed. As-prepared functional microgels (TR/Au NPs/MGs) allow digestion of Cyt c within 15 s under microwave irradiation. The internal standard SALDI-MS approach provides linearity (R(2) = 0.98) of MS signal ratio (I 1168.6/I 1067.6) of the tryptic digested peptide (m/z 1168.6) to GR-10 peptide (m/z 1067.6) against the concentration of Cyt c ranging from 25 to 200 nM, with a limit of detection (at a signal-to-noise ratio of 3) of 10 nM. This approach has been validated by the analysis of the lysates of HeLa cells, with an average concentration of 13.7 ± 3.5 μM for cytoplasmic Cyt c. Increased concentrations of Cyt c in the HeLa cells treated with etoposide (a commercial drug) or carbon dots (potential drug) have been revealed through this simple, sensitive, and rapid SALDI-MS approach, supporting the drugs induced Cyt c-mediated apoptosis of the cells. This study has shown that this internal standard SALDI-MS approach holds great potential for cell study.