Exploration of microbial dynamics of Streptomyces lavendulae ACR-DA1, a psychrotrophic isolate from the North-Western Himalayan cold desert, was carried out using matrix-assisted laser desorbtion ionisation-time of flight mass spectrometer. Valinomycin was found as a major produce and cyclic depsipeptide montanastatin as a minor produce. The yield of the valinomycin was found to be 0.3 mg l-1 in submerged growth condition at the batch scale. Miniaturization of optimization experiments was adept to maximize the production using the expeditious and efficient technique of intact cell mass spectrometry. The present study showed that using optimized conditions and growing the culture in synthetic mineral base starch medium at 10 °C enhanced the production to 19.4 mg l-1. Our results demonstrated 64-fold increase in yield from the wild-type S. lavendulae ACR-DA1 strain using a simple and economical downstream process.