Background In addition to increasing linear growth, improvement in health-related quality of life (HRQOL) is an important endpoint in the treatment of short statured youth. Hence, condition-specific psychometric valid instruments that adequately assess HRQOL are needed. We aimed to confirmatorily examine the psychometric performance of the Quality of Life in Short Stature Youth (QoLISSY) questionnaire used in a previously reported prospective randomized open-label trial. Methods This trial compared treatment of idiopathic short stature (ISS) in 76 adolescent males with either oral aromatase inhibitors (AIs), subcutaneous daily growth hormone (GH) or a combination treatment (AI/GH) for at least 2 years, demonstrating improvements in HRQOL with the GH and AI/GH interventions. HRQOL was assessed from the child’s and parent’s perspectives with the short stature-specific QoLISSY and the generic KIDSCREEN questionnaires before and 24 months into treatment. Scale scores and psychometric properties were examined regarding reliability and validity of the QoLISSY questionnaire using the dataset from the published trial. Results The QoLISSY questionnaire showed high internal consistency and satisfactory criterion, convergent and known-groups validity. Scale scores were evenly distributed with no major floor or ceiling effects. Responsiveness analyses suggest that the QoLISSY questionnaire detects significant changes in HRQOL after 2 years of treatment with growth-promoting therapies in children with short stature from both the child’s and parent’s perspectives. Conclusions The QoLISSY questionnaire is a psychometrically sound, reliable and valid instrument that can explore the experiences associated with short stature, track HRQOL changes over time and in response to treatment, and highlight HRQOL domains that can be improved through intervention.