Well-being is a multifactorial positive state that is highly influenced by some endogenous molecules that control happiness and euphoric feelings. These molecules, e.g., neurotransmitters, hormones and their derivatives, play a crucial role in metabolism and may be referred to as “well-being-related markers”. The deregulation of well-being-related markers can lead to organism malfunctions and life-threatening states. In this research, we aimed to evaluate the potential of nails for the chronic production of several well-being-related markers. For this purpose, we developed an LCMS /MS-based method for the determination of 10 well-being-related markers, including melatonin, serotonin, cortisol, kynurenine and several precursors and metabolites. The method was optimized regarding different analytical steps: required sample amount, extraction time, number of required extractions, preconcentration, injection volume and MS conditions. Method validation was performed by two different approaches: (i) using surrogate nail matrix and (ii) using authentic nail samples by standard additions. The method was found to be linear in the expected endogenous range and sensitive enough to determine the low endogenous concentration levels in nails. Accuracy and precision were appropriate in both validation approaches. As proof of concept, the method was used (i) to correlate fingernail and toenail levels for all metabolites in 22 volunteers, (ii) to establish the endogenous concentration range of all metabolites in females (n = 50) and males (n = 34) and (iii) to correlate the metabolite levels with age. For some metabolites, the calculated ranges have been reported for the first time. In summary, the present strategy to evaluate well-being-related markers in nails may be a useful tool for the evaluation of the production of these important compounds with high potential for a wide range of clinical purposes.