Pain management is a general concern for healthcare quality. In the particular context of neonatal care, it\'s well known that an efficient pain management will decrease mortality and morbidity of newborn infants. Furthermore, the plasticity of developing brain is vulnerable to pain and/or stress, that in turn may cause long term neurodevelopmental changes, including altered pain sensitivity and neuroanatomic and behavioural abnormalities. During neonatal intensive care stay, large number of painful procedures are performed, the majority of which are not accompanied by adequate analgesia. Optimal management requires competent pain assessment which can be especially difficult to perform in this non verbal population. We have developed an instantaneous heart rate variability (HRV) analysis method, non intrusive and user-friendly, based on the ECG signal acquisition. This analysis method enabled us to design parameters related to the influence of pain on the Autonomic Nervous System (ANS) activity. This paper presents the application of this method, previously validated for adults under general anesthesia, to the domain of newborn infants prolonged pain assessment.