Background: Salivary cortisol measurement is a non-invasive method suitable for use in neonatal research. Mother-infant separation after birth represents stress and skin-to-skin contact (SSC) has numerous benefits. The aim of the study was to measure salivary cortisol in mothers and newborns before and after SSC in order to assess the effect of SSC on mothers' and infants' stress and to estimate the efficacy of collecting small saliva samples in newborns. Methods: Salivary cortisol was measured in 35 mother-infant pairs before and after the first and the fifth SSC in small saliva samples (50 mL) using the high sensitivity Quantitative ELISA-Kit (0.0828 nmol/L) for low cortisol levels detection. Samples were collected with eye sponge during 3 to 5 minutes. Results: Cortisol level in mothers decreased after SSC: the highest levels were measured before and the lowest after SSC and the differences in values were significant during both the first (p<0.001) and the fifth SSC (p<0.001). During the first SSC the cortisol level decrease was detected in 14 (40%) and an increase in 21 (60%) newborns, and during the fifth SSC a decrease was detected in 16 (45.7%) and an increase in 19 (54.3%) newborns, without confirmed significance of the difference. Saliva sampling efficacy using eye sponge was 75%. Conclusions: Cortisol level decrease in mothers proves the stress reduction during SSC, while variable cortisol levels in infants do not indicate stress reduction and imply the need for further research. The used sampling method appeared to be one of the most optimal considering the sample volume, sampling time and efficacy.