Background: Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models. Methods: We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and PCR. Results: Analyzing a 50L dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood-spots from two patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites. Conclusion: Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.