By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto metal electrodes. Previous studies have shown that the electron transfer rate through the protein is a function of the position of this heme group with respect to the electrode surface. In this study a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH3(+)) and carboxyl (COO(-)) functionalized gold is presented. Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by x-ray photoelectron spectroscopy. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well ordered film. Time of flight secondary ion mass spectrometry data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC (cysteine: C2H6NS(+); glutamic acid: C4H6NO(+) and C4H8NO2 (+); leucine: C5H12N(+)). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 50 % increase in this ratio, observed between the protein covered NH3 (+) and COO(-) substrates, indicates opposite orientations of the CytoC on the two different surfaces.