Wearable healthcare technologies should be non-invasive, robust to daily activity/environments, easy to use, and comfortable to wear. Flexible substrate devices for biomarker monitoring can contribute to wearable diagnostic applications. Single-target biosensors have extensively been developed for health-monitoring applications; however, recently multiplex biomarker tests have generated clinical interest. Targeting multiple biomarkers in diagnostic systems (wearable or point of care) offers more focused diagnosis and treatment as changes in a single biomarker can be caused by a series of physiologic conditions. This review highlights flexible substrates that have been successfully demonstrated for multiplex biomarker detection with potential for healthcare monitoring.