Ascidians (Tunicates/Urochordates) are excellent animal models for developmental, evolutionary and molecular biology studies. Isolation of DNA is the basic phase for most of the molecular biology studies including DNA barcoding. So far there is no definite method for the isolation of DNA from ascidians. In an effort to overwhelm this problem, three different methods such as Phenol-Chloroform, Salting out and Kit method were evaluated for DNA extraction. Results showed that among the three methods, kit method, after few modifications, yielded sufficient quantity and quality of DNA. The yield of DNA between the samples and methods was significant (P<0.005). This method is more pronounced for the PCR amplification of COI gene with tunicate primer.