Multiple sequence alignment (MSA) has assumed a key role in comparative structure and function analysis of biological sequences. It often leads to fundamental biological insight into sequence-structure-function relationships of nucleotide or protein sequence families. Significant advances have been achieved in this field, and many useful tools have been developed for constructing alignments. It should be stressed, however, that many complex biological and methodological issues are still open. This chapter first provides some background information and considerations associated with MSA techniques, concentrating on the alignment of protein sequences. Then, a practical overview of currently available methods and a description of their specific advantages and limitations are given, so that this chapter might constitute a helpful guide or starting point for researchers who aim to construct a reliable MSA.