The present invention describes a process for isolation of romidepsin from fermentation broth and preparation of crystals of romidepsin. The process of the invention includes fewer purification steps and provides romidepsin having purity of greater than 99.5% area by HPLC. The process of the invention involves simple purification steps and hence, does not require multiple chromatographic purification steps to achieve desired quality of romidepsin. The process is advantageous over reported processes in terms of 99.5% pure yield, fast process, less expensive and less cumbersome as multiple chromatographic purification is not necessary to achieve desired quality. The process for the preparation of crystals of romidepsin provides advantages like simple steps and involves use of single solvent. The process is advantageous in terms of time, cost, and simplicity.