Under field conditions, most of the broiler chicks receive feed and water 24-36 hours after hatching results in decreases body weight and impairs overall performance. Hence, a biological trial was carried out in broilers to investigate the effect of In-ovo injection of glucose, lysine, threonine and β-hydroxy-β-methylbutarate (HMB) on the morphology of digestive organs in commercial broilers. On the 18th day of incubation, of total 540 embryonated, each 90 eggs were injected with 0.5 ml of 10 % glucose solution (T3), 0.5 ml of 0.5 % lysine solution (T4), 0.5 ml of 0.5 % threonine (T5), 0.5 ml of 0.5 % β-hydroxy-β-methylbutarate (HMB) (T6) along with 90 eggs as non-injected control (T1) and 90 eggs as injected control. A pin head size hole was made just below the air cell and 0.5 ml of (In-ovo injection) nutrient solution was injected into the amnion using an insulin syringe. The hole was sealed and the period of incubation was completed. Biological feeding trial was continued up to 42 days with 288 broiler chicks from different In-ovo treated groups each with three replicates of sixteen chicks each. The chicks were fed ad libitum with broiler pre-starter, starter and finisher mash as per BIS (2007) specifications from 1-10, 11-21 and 22-42 days of age respectively. Data on absolute weight of digestive organs at sixth week of age was recorded by using an electronic balance with 0.2 g accuracy and subjected to statistical analysis. Results revealed that In-ovo injection of glucose, lysine, threonine and HMB on 18th of incubation produced significantly heavier liver and pancreas on absolute weight basis. However, other digestive organ absolute and relative weights were not significantly influenced in this study. In-ovo injection of vital nutrients increased only accessory digestive organs like liver and pancreas of broilers whereas other digestive organs weight was not affected.