Sub clinical mastitis is responsible for heavy economic losses throughout the world in dairy sector. The objective of present study was to investigate lactate dehydrogenase (LDH) enzyme activity in milk for detection of subclinical mastitis in buffaloes Milk samples from 60 buffaloes, including 40 subclinical infected and 20 healthy were subjected to the estimation of LDH using a LDH activity assay kit. LDH concentration in milk of buffaloes having subclinical mastitis was reported to be high (1.88±0.91 µmole/ml/min) as compared to healthy animals (0.26±0.10 µmole/ml/min). The receiver operating characteristics analysis at cut off value of 0.44 µmole/ml/min for LDH activity exhibited sensitivity and specificity of 95% each and area under curve was 0.959. Results of the correlation matrix revealed significantly strong positive interdependence of somatic cell count with enzyme LDH (r = 0.722**; P <0.01). Measuring LDH activity in milk was found both easy and cost effective with high sensitivity and specificity indicating that LDH activity has great potential as a diagnostic tool for detection of sub clinical mastitis in buffaloes.