Objective: To determine pain response of infants to change in sequence of Pentavalent and PCV vaccine injection. Design: Single-center, randomized, parallel group, active controlled trial Setting: Immunization clinic, Department of Pediatrics, LLRM medical college, Meerut, India. Participants: Healthy full term infants upto 4 months of age. Intervention: Infants received either pneumococcal conjugate vaccine(PCV) or Pentavalent vaccine first, followed one minute later by the other vaccine. Infants were videotaped from 5 to 10 seconds prior to first vaccine injection till 3 minutes after second vaccination using a handheld video camera. The primary outcome measure was infant pain assessed by modified facial coding system (MFCS) and neonatal infant pain scale (NIPS) from the videotapes by research assistants. Results: 130 participants were enrolled in study after consent 65 in each group. Outcome data for 128 infants were available for primary outcome analysis because of missing data of 2 infants in group A. Pain scores, MFCS and NIPS per infants were significantly lesser in group B than group A upto 1 minute after second vaccination. Similarly lesser change in hemodynamic parameter like heart rate and SpO2 was observed in group B. Total cry duration was also observed lesser in group B. Conclusion: Simple change in order of vaccine is an effective non-pharmacological intervention in reducing pain during vaccination of infants. Infants experience lesser pain when Pentavalent vaccine is given first followed by PCV, instead when PCV vaccine is given first. Keywords: PCV (Pneumococcal conjugate vaccine), SpO2 (Oxygen saturation), MFCS and NIPS, Infant pain, Pentavalent vaccine.