The combination of tandem spectrometry and database searching is one of the most popular technologies for protein identification. However, only those proteins in the searching database could be identified, and current database is far from completeness. So it is necessary to mining the MS/MS data comprehensively, in which novel protein identification is the most important one. The definition of novel protein could be divided into three levels according to their annotations of sequences and functions. As a part of protein identification, the main approaches used to identify novel protein are basing on the following two different ways: de novo sequencing combined with similarity search and searching against nucleotide acid databases such as EST or genome databases. Several mature or newly developed methods and techniques were summarized, and the problems and strategies discussed here would be helpful for the related researches.