Acute mental stress has become a pervasive issue in modern societies. Excessive exposure can have far-reaching consequences for well-being and health and is a recognized but underappreciated risk factor. The concept of stress assessment, as presented in this work, helps to overcome typical misconceptions of “stress measurement” and to identify levers to promote well-being. Although numerous measures have been proposed to quantify the effects of acute mental stress, a methodological standard remains elusive. Non-invasive physical measures that have been used to detect effects of stress (strain) target skin conductance, heart rate variability, QT variability, hemodynamics, respiration, and cortisol concentration. These measures are linked to physiological effects of acute mental stress mediated by activation of the autonomic nervous system and the hypothalamic-pituitary-adrenal axis. Effective stress assessment requires careful consideration of measure sensitivity, specificity, methodological requirements, and instrumental requirements. This review aims to inform and guide researchers and practitioners to support the development of effective stress assessment procedures and interventions that promote well-being and improve quality of life.