Purpose <p>Diet quality has been associated with health-related quality of life (HRQoL) in adolescents, but the role of specific nutrient substitutions, particularly between protein sources, remains unclear. This study aimed to examine the association between isocaloric substitution of plant-based and animal-based protein and HRQoL in adolescents, including total and food-group-level substitution analyses.</p> Methods <p>This cross-sectional study used data collected in the Eating Healthy and Daily Life Activities (EHDLA) study (2021–2022, Murcia, Spain) in 617 adolescents aged 12–17 years. Total and food-group-level isocaloric substitution analyses were performed using multivariate nutrient density models (% energy; 5% substitution). HRQoL was assessed with KIDSCREEN-10 (0–100 points). Models were adjusted for age, sex, body mass index (z-score), socioeconomic status, total energy intake, sleep duration, physical activity, and sedentary behaviour.</p> Results <p>Among 617 adolescents (57.1% girls; median age 14.0 years [interquartile range (IQR): 13.0–15.0]), isocaloric substitution of plant-based protein for animal-based protein was associated with higher HRQoL (standardized beta coefficient [<i>β</i>] = 0.37 standard deviation [SD] units; <i>p</i> &lt; 0.001). In food-group analyses (<i>β</i> in SD units), the strongest positive associations were observed when substituting plant-based protein for protein from poultry (<i>β</i> = 0.44) and red meat (<i>β</i> = 0.43). Substituting animal-based protein for protein from legumes (<i>β</i> = −0.64) and nuts (<i>β</i> = −0.56) was associated with lower HRQoL.</p> Conclusions <p>In the present study, the substitution of plant protein for animal protein was generally associated with higher HRQoL scores in adolescents. Further longitudinal and experimental studies are needed to confirm these associations and clarify underlying mechanisms.</p>