Background: Assessing pain in mechanically ventilated infants is challenging. The assessment of skin conductance (SC) is based on the sympathetic nervous system response to stress. This study purpose was to evaluate the validity of SC for assessing pain in mechanically ventilated infants. Methods: A prospective cross-sectional observational design was used to study SC and its relation to: the category of procedure (i.e., painful or non-painful); the phase of procedure (i.e., before, during and after), and referent pain measurements (i.e., Premature Infant Pain Profile-Revised (PIPP-R) and Neonatal Facial Coding System (NFCS)). Eligible infants were those up to 12 months of age, in intensive care units, who were mechanically ventilated, and required painful and non-painful procedures. Results: From October 2017 to November 2018, 130 eligible infants were identified, and 55 infants were studied. SC (number of waves per second) during painful procedures (median 0.27, interquartile range 0.2-0.4) was statistically significantly higher than those during non-painful procedures (0, 0-0.09). SC during painful procedures was statistically significantly higher than those before (0, 0-0.07) and after painful procedures (0, 0-0.07). SC showed moderate statistically significant positive correlations with PIPP-R (Spearman's rho = 0.4-0.62) and the four-item NFCS (Spearman's rho = 0.31-0.67) before, during and after painful or non-painful procedures respectively. SC had excellent performance (area under the receiver operator curve = 0.979) with excellent sensitivity (92.31%), specificity (95.42%) and negative predictive value (99.21%) but only sufficient positive predictive value (66.67%) when used to discriminate moderate-to-severe pain. Conclusions: SC showed good validity for assessing pain in critically ill infants requiring mechanical ventilation. Significance of the study: Pain assessment in mechanically ventilated infants is challenging. In this study, the validity of skin conductance (SC) for pain assessment is evaluated in the same population of infants during painful and nonpainful procedures. SC showed good validity for assessing acute pain in relation to category of procedure, phase of procedure, and referent pain measurements. SC is a promising method, especially with other pain assessment methods and other determinants of 1995