Researchers and clinicians are becoming increasingly aware of the importance of assessing positive functioning to inform clinical outcomes. This paper evaluates the Questionnaire on Well-Being (QWB, available for free https://doi.org/10.17605/OSF.IO/GSC3R), a clinically informed instrument that assesses subjective well-being, across two studies. Study One, consisting of treatment-seeking individuals in an assertiveness training sample (n = 495), explored the factorial structure of the QWB, assessed the four-week test-retest reliability, criterion-related validity, and identified a preliminary cutoff point for the QWB with clinical significance. Study Two, including participants from the general public (n = 1561), confirmed the factorial structure of the QWB and further evaluated criterion-related validity. The results provided support for a unidimensional structure for the QWB. Furthermore, the QWB exhibited excellent internal reliability (Cronbach’s alpha = 0.93 and 0.94 in Study One and Two, respectively), high test-retest reliability (ICC₃ = .50 at a four-week follow-up in Study One), and appropriate criterion-related validity demonstrating positive correlations with positive affect and negative correlations with psychopathology. Finally, a cutoff point on the QWB below 50 was associated with marked psychopathology. These findings provide preliminary support for the usage of the QWB in clinical and non-clinical settings, establishing the QWB as a reliable indicator of subjective well-being.