Managing pain is critical, especially for premature infants undergoing frequent painful procedures. Uncontrolled pain can lead to lasting harm in growth, cognitive development, and future pain responses. A double-blinded clinical investigation involving 150 premature infants was performed in a neonatal intensive care unit. They were randomly divided into three groups: Sucrose 20% (50 infants), distilled water (50 infants), and a control group (50 infants). The infants' behavioral responses were assessed using an infant pain measurement tool before, at 2, and 7 min after the intervention through direct observation. The study revealed that mean pain scores before, 2 min after, and 7 min after the intervention in the sucrose group were (4.78±0.91), (3.18±1.15), and (2±1.02), respectively. In the distilled water group, scores were (4.66±0.89), (3.04±1.15), and (3.08±1.10), while in the control group, they were (4.0±0.79), (4.94±0.79), and (4.72±0.96). The trend of pain scores varied among the groups over time, with a significant difference in mean pain scores at different time points (P<0.001). Initially comparable, pain scores notably decreased after 2 min in the sucrose and distilled water groups (P<0.001), differing from the control group. The study indicated that 20% sucrose and distilled water equally reduce infant pain post-venipuncture, suggesting their viability for clinical pain management. Distilled water, however, provides additional benefits, including economic considerations and ease of preparation.