The masquerade attack, where an attacker takes on the identity of a legitimate user to maliciously utilize that user’s privileges, poses a serious threat to the security of information systems. Such attacks completely undermine traditional security mechanisms due to the trust imparted to user accounts once they have been authenticated. Many attempts have been made at detecting these attacks, yet achieving high levels of accuracy remains an open challenge. In this paper, we discuss the use of a specially tuned sequence alignment algorithm, typically used in bioinformatics, to detect instances of masquerading in sequences of computer audit data. By using the alignment algorithm to align sequences of monitored audit data with sequences known to have been produced by the user, the alignment algorithm can discover areas of similarity and derive a metric that indicates the presence or absence of masquerade attacks. Additionally, we present several scoring systems, methods for accommodating variations in user behavior, and heuristics for decreasing the computational requirements of the algorithm. Our technique is evaluated against the standard masquerade detection dataset provided by Schonlau et al. [Schonlau, M., DuMouchel, W., Ju, W.H., Karr, A.F., Theus, M., Vardi, Y., 2001. Computer intrusion: Detecting masquerades. Statistical Science 16 (1), 58–74], and the results show that the use of the sequence alignment technique provides, to our knowledge, the best results of all masquerade detection techniques to date.