The present study reports the determination of optimum thawing temperature and holding period of the Mareks Disease (MD) cell associated vaccine after reconstitution by plaque assay. The highest titre of MD HVT+SB-1 bivalent vaccine was obtained at the thawing temperature of 35°C for 45 sec followed by 26°C for 45 sec, 20°C for 60 sec and 40°C at 45 sec; when the vaccine was held on ice after reconstitution. The difference in virus titre among 30, 90 and 120 min holding period at 26°C and 35°C thawing for 45 sec was significant (P≤0.001). However the variation in titre between 30 and 90 min holding period was not significant for thawing temperatures of 20°C and 40°C (P>0.05). No plaques were seen when the bivalent HVT+SB-1 vaccine was thawed at 45°C for 45 sec. The titre obtained at 40°C was extremely low after a holding period of 120 min. After thawing at 35°C for 45 sec and when the reconstituted vaccine was held at RT, there was a drastic reduction in virus titre by 120 min. The study revealed that thawing at 20°C and 40°C for 45 sec would severely lower the titre of HVT+SB-1 vaccine. The complete loss of virus titre was seen at thawing temperature of 45°C for 45 sec. The study reasserts the importance of maintaining the cold chain, appropriate thawing temperature and holding period after reconstitution of HVT+SB-1, a cell associated MD vaccine in maintaining the requisite PFU/dose of vaccine.