Depth profile analyses of ultra-thin layers and their interfaces are investigated, using an innovative Pulsed-radiofrequency Glow Discharge Time-of-Flight Mass Spectrometer (pulsed-rf-GD-TOFMS), to obtain information about the presence of minor elements and to evaluate segregation/diffusion processes at the interfaces. A series of ultra-thin Nb/Al1−xCox bilayers, deposited on Si wafers by dc-magnetron sputtering, is analyzed. An Al1−xCox layer is first deposited on the Si substrate with controlled constant thickness (tAlCo = 6 nm), and different stoichiometries (Co atomic concentration, x = 0, 0.015, 0.035, 0.045, 0.09, 0.35). Then, a Nb layer is deposited on top of the AlCo one, with a thickness tNb = 50 nm that is also kept constant along the whole series. Qualitative depth profiles of those layered sandwich-type samples are determined using our pulsed-rf-GD-TOFMS setup, indicating segregation of Al and Co and diffusion of Co into the Si substrate, as a function of the Co atomic concentration in the AlCo internal layer. Additionally, a linear calibration curve was achieved plotting the Co atomic concentration versus the total integrated Co ion signal, allowing the Co quantification in ultra-thin layers (∼6 nm). Moreover, Secondary Ion Mass Spectrometry (SIMS) was used as a reference technique to validate the high analytical potential of pulsed-rf-GD-TOFMS for the analysis of ultra-thin layers and interfaces. The comparative analytical performance of both techniques is discussed.