The analysis of blood-derived circulating tumor DNA (ctDNA) enables minimally-invasive molecular screening for the clinical guidance of cancer patients. However, ctDNA comprises only a small fraction of the total DNA in blood and therefore highly sensitive techniques are required for reliable detection. We developed a robotized hybrid capture-based NGS assay using Twist probes and bioinformatic analysis pipeline for the detection of ctDNA from plasma focusing on actionable targets in signal transduction pathways. To assess the assays performance, we used ctDNA mimicking reference standards and plasma of healthy individuals.