Background The Antidepressant Medications: Biology, Exposure & Response (AMBER) research programme was established to investigate the biological mechanisms underlying antidepressant action and variability in treatment response. Generation Scotland holds detailed genomic, clinical, and health information with recontacting consent, making this cohort ideal for investigating these aims. Methods We deployed a questionnaire, developed with input from a Lived Experience panel, to the Generation Scotland cohort to gather data on their depressive symptoms, medication history, efficacy, and side effects to develop clinically meaningful phenotypes of antidepressant response. Invitations were sent to 15,117 Generation Scotland participants who were 18 years or older and consented to be recontacted. Between July and November 2025, 1,180 participants with a history of antidepressant treatment for depression completed the questionnaire. Results The sample was predominantly female (78.1%), self-identified as White (98.6%), and older (median age 57 years) than the wider Generation Scotland cohort (median 49 years) and Scottish population (median 41.3 years). Participants reported heterogeneous depressive symptom profiles spanning mood, anxiety, cognitive, sleep, behavioural, and physical domains. One-third of participants (31.1%) had taken three or more different antidepressants. Selective serotonin reuptake inhibitors (SSRIs) were the most common class (89.1%). Using self-reported treatment duration, discontinuation patterns, and efficacy, we developed a stringent classification system to capture treatment response extremes, where 23.8% were classified as responders and 1.5% as non-responders, with the majority unclassified. Conclusions Questionnaire data will be linked with electronic health records to validate antidepressant response classifications. Following validation, 25 responders and 25 non-responders will provide biological samples for DNA methylation profiling and generation of patient-derived cell lines. These models will be exposed to SSRIs to identify gene expression signatures and biological pathways distinguishing treatment response, integrating with genomic and clinical data across the AMBER project. These findings will provide a valuable resource for future antidepressant response research.