Evaluating the mole fraction of hydrogen isotopes in a solid is a difficult task. Few methods allow it to be achieved. LIBS is a laser method based on the electronic excitation of elements and the spontaneous emission of characteristic optical lines. On a sample containing hydrogen isotopes, α-type lines can allow the estimate of their total mole fraction. In addition, LIBS is a discriminating method because it can separate the contributions of isotopes. This paper reports the implementation of this method on thin film-type samples containing hydrogen and tritium.They consist of nanometric layers of palladium and titanium on a silicon substrate. Under irradiation of nanosecond laser pulses reaching a fluence of the order of 200 J cm−2, LIBS was performed in argon at atmospheric pressure. A detailed spectroscopic study is performed around the Tα line of wavelength 656.039 nm of the Balmer series (n=3→2) of tritium. An analysis based on the reconstruction of the spectrum under conditions of local thermodynamic equilibrium is carried out. This leads to the estimate of the tritium mole fraction.