The present study was conducted to investigate biochemical analytes during different stages of lactation in Surti buffaloes. 10 healthy Surti buffaloes were selected at postpartum (n=10). Blood samples from all the animals were collected at early (30±5 days), mid (100±5 days) and late lactation (200±5 days) and serum was obtained and analysed for various biochemical metabolites. Serum biochemical metabolites such as total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, total protein, albumin and globulin increased significantly (p≤0.05) whereas urea and creatinine significantly (p≤0.05) decreased from early to late stages of lactation. It is thus concluded that biochemical metabolites such as total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, total protein, albumin and globulin increased whereas urea and creatinine decreased from early to late lactation. These changes suggest close monitoring of these metabolites in Surti buffaloes during different stages of lactation for nutritional intervention if required.