Human neutrophil peptides (HNPs) and the human cathelicidin LL-37 are antimicrobial peptides secreted by neutrophils, which play a crucial role in innate immune responses. The aim of this study was to establish a new method for ProteinChip arrays in combination with surface enhanced laser desorption/ionization (SELDI) technology and time-of-flight mass spectrometry to analyze gingival crevicular fluid (GCF) samples. To optimize experimental conditions, four different ProteinChip arrays (NP20; CM10, pH 4; CM10, pH 7; IMAC) along with corresponding binding buffers were tested. GCF samples were collected from patients showing healthy periodontal sites and sites with early signs of inflammation (gingivitis), but with no pocket depth greater than 4 mm. For GCF analysis, NP20 arrays and CM10 (pH 4) arrays showed specific and reproducible profiles in the range of 2.5-30.0 kDa. Donors that demonstrated significantly higher intensity peaks corresponding to the mass of LL-37 (p=0.01) also tended to show greater intensity peaks corresponding to the masses of HNP-1 and HNP-2 in samples from inflamed compared to healthy periodontal sites. The findings indicate that analysis of GCF samples by SELDI-TOF mass spectrometry is a useful approach to simultaneously analyze multiple markers, such as antimicrobial peptides, which may be beneficial for determination of new periodontal risk factors.