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Lupin alkaloids (LAs) represent a class of toxic secondary metabolites in plants, in particular in <i>Lupinus</i> spp.; they are produced as a defense mechanism due to their strong bitter taste and are very dangerous for human and animals. In this work, a sensitive and reliable high performance liquid chromatography—tandem mass spectrometry (HPLC-MS/MS) analytical method for the identification and quantification of thirteen lupin alkaloids was developed and validated according to FDA guidelines. Efficient extraction and clean-up steps, carried out by solid-phase extraction, were finely tuned on the basis of the characteristics of the analytes and lupin samples, providing good selectivity with minimized matrix interference. The effectiveness of the method was proven by the satisfactory recovery values obtained for most of the analytes and a matrix effect ≤23% for all tested levels. In addition, a sensitive and reliable determination of the target compounds was obtained; LOQs were between 1 and 25 µg Kg⁻¹, i.e., below the requested maximum levels (<200 mg Kg⁻¹). The method was applied to evaluate the LAs profile in different batches of raw <i>L. albus</i> L. samples, varying in size and across farming treatments.