Spectral fingerprints were acquired for Rio Red grapefruit using flow injection electrospray ionization with ion trap and time-of-flight mass spectrometry (FI-ESI-IT-MS and FI-ESI-TOF-MS). Rio Red grapefruits were harvested 3 times a year (early, mid, and late harvests) in 2005 and 2006 from conventionally and organically grown trees. Data analysis using analysis of variance principal component analysis (ANOVA-PCA) demonstrated that, for both MS systems, the chemical patterns were different as a function of farming mode (conventional vs organic), as well as growing year and time of harvest. This was visually obvious with PCA and was shown to be statistically significant using ANOVA. The spectral fingerprints provided a more inclusive view of the chemical composition of the grapefruit and extended previous conclusions regarding the chemical differences between conventionally and organically grown Rio Red grapefruit.