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Objective This study was designed with objective to study pain response of infants to change in sequence of administration of Hepatitis B and DTwP vaccines. Methods This was a randomized parallel control trial. The study was carried out in the immunization clinic of the Department of Pediatrics, LLRM Medical College, Meerut. One hundred and thirty healthy term infants up to 4 months of age were injected either DTwP vaccine first or Hepatitis B vaccine first, followed one minute later by the other vaccine. Result Baseline characteristics did not differ between the groups. The mean (SD) of AUC of MFCS and NIPS was significantly more in DF group as compared to HF group (for MFCS 25.5 ± 5.4 versus 22.5 ± 5.5, p < 0.01; for NIPS 31.77 ± 5.5 versus 27.64 ± 6.9, p < 0.01). Similarly mean (SD) of AUC of Heart rate and saturation of oxygen showed significant variation in DF group as compared to HF group (for heart rate 591.6 ± 55 versus 559.6 ± 49, p < 0.01; for SpO 2 326.4 ± 12 versus 335 ± 8, p < 0.01). Conclusion These results showed that infant experienced lesser pain when Hepatitis B was administered first than when DTwP vaccine was given first. [ABSTRACT FROM AUTHOR]