Quantification of rumen microbial biomass synthesis assumes significance as rumen fermentation of feed organic matter and microbial biomass synthesis meet 70-85% of energy and 70-100% of protein needs of ruminants. Therefore, it is important to understand the feed factors influencing higher microbial biomass in the rumen. Determination of feed factors affecting microbial biomass synthesis in vivo is expensive. Hence, the rumen in vitro gas production technique used for feed energy evaluation in ruminant feedstuffs was adopted to assess microbial biomass production potential of feedstuffs based on the concept of partitioning of fermented organic matter between microbial biomass and fermentation waste products. Therefore, in this study, diets were formulated to differ in differ in microbial biomass synthesis potential as partitioning factor (PF) based on in vitro rumen fermentation characteristics and tested them in vivo by measuring feed intake, nutrient digestibility, nitrogen balance, purine excretion in urine and production performance in growing lambs and lactating cows.