The present study was designed to examine the influence of varying levels of limiting amino acids (lysine, methionine and threonine) supply to lactating Nili-Ravi buffaloes on their nutrient’s intake and utilization, milk yield and composition, nitrogen balance and hematological parameters. Fifteen lactating Nili-Ravi buffaloes in mid lactation with almost similar milk production were randomly divided into three different groups of five females within each according to completely randomized design. Three iso-caloric and iso-nitrogenous diets with high, medium and low levels of lysine, methionine and threonine concentrations (% of crude protein) were formulated and represented as high essential amino acids (HEAA), medium essential amino acids (MEAA) and low essential amino acids (LEAA) concentrations, respectively. The study lasted for 100 days; first ten days were given for the adaptation to new diets while every six days after every month of the remaining period served as collection periods. The intake (% body weight) of dry matter (DM), crude protein (CP), metabolizable energy (ME), neutral detergent fiber (NDF) and acid detergent fiber (ADF) did not show any treatment effect. However, the intake of rumen un-degradable protein, lysine, methionine, and threonine reduced linearly (P<0.01) with decreasing concentrations of these nutrients in the experimental diets. A linear increase (P<0.01) in DM, NDF and ADF degradabilites was recorded with gradually decreased dietary RUP and EAA concentrations while CP digestibility remained unchanged. Milk yield and daily weight gain decreased linearly (P<0.01) with reducing dietary levels of EAA concentrations. A positive nitrogen balance was also observed in buffaloes fed all the experimental diets but it was similar statistically. Hematological analysis indicated decreased (P<0.01) mean corpuscular volume and hemoglobin concentrations in buffaloes fed gradual decreased dietary EAA concentrations. It is concluded that the varying levels of limiting amino acids have effect on nutrient intake, utilization, milk yield and hematological parameters. These results could be used for further studies of this field.