Background: Acquisition of point mutations in KRAS gene is causally associated with the onset of development of a resistance to anti-EGFR therapy in colorectal cancer. Newly acquired KRAS mutations can be detected in blood plasma months before radiographic detection. The objective of this study was to demonstrate feasibility of an ultrasensitive non-invasive method for detection of KRAS mutations in urine and plasma of patients with advanced colorectal cancer. Methods: Archived, matched urine and plasma samples (stored between 3-5 years prior to ctDNA extraction) from 20 treatment naïve, advanced stage cancer patients with known tumor tissue KRAS mutations determined by an accredited clinical laboratory, were used in a retrospective setting for a blinded concordance study. KRAS status in urine and plasma was compared to that in tumor tissue in order to assess clinical sensitivity of the ctDNA assay. An ultrashort-amplicon (31bp) assay for KRAS mutation enrichment and detection in highly fragmented urinary and plasma ctDNA was developed. The assay detected 1 copy of KRASG12A/C/D/R/S/V or G13D mutant allele in a background of wild-type DNA with a verified analytical sensitivity of 0.007% (7 copies per ~100,000 genome equivalents). Results: In a pilot study of 20 advanced stage colorectal cancer patients, 15 of 16 evaluable archived urine samples (94%) had KRAS mutation that was concordant with tissue biopsy. Of 20 archived plasma samples evaluated, 19 (95%) displayed the KRAS mutation concordant with tumor tissue. Of 16 paired urine and plasma samples, 15 (94%) had concordant KRAS mutation calls. Conclusions: This study demonstrates high clinical sensitivity (≥94%) of concordant KRAS mutation detection between urine, plasma and tissue specimens from advanced colorectal cancer patients. Early detection and monitoring of acquired KRAS mutations in circulating tumor DNA, and in particular urinary ctDNA, opens the possibility of a new paradigm for a truly non-invasive method of individualized care for colorectal cancer patients.