Barbiturates are highly susceptible to dissociation in mass spectrometry (MS) because of their long side chains combined with a nonaromatic ring consisting of several carbonyl and amine groups. As a result, they exhibit extensive α-cleavage and subsequent rearrangement, making the identification of these compounds difficult. Although a library of electron ionization MS (EIMS) is available, most barbiturates have very similar fragment patterns. Accordingly, it would be desirable to develop a technique for soft ionization, providing a molecular ion and large fragment ions as well. In this study, a molecular ion was clearly observed, in addition to large fragment ions, for a variety of barbiturates based on multiphoton ionization MS (MPIMS) using a tunable ultraviolet femtosecond laser as the ionization source (fs-LIMS). This favorable result was achieved when the optimal laser wavelength for minimizing the excess energy remaining in the ionic state was used. An examination of the photofragmentation pathways suggested that an H atom in the side chain was abstracted by an oxygen atom in the carbonyl group in the ring structure thus initiating fragmentation and subsequent rearrangement. Barbiturates that are substituted with alkyl groups (amobarbital and pentobarbital) had narrower spectral regions for optimal ionization than the other barbiturates with alkyl and alkenyl groups (butalbital and secobarbital) and more with alkyl and phenyl groups (phenobarbital). All of the barbiturates studied provided unique mass spectral patterns in fs-LIMS, which was useful for the reliable identification of these compounds in practical trace analysis.