AminoIndex Cancer Screening (AICS) is a novel cancer screening test based on plasma free amino acid (PFAA) levels. This system categorises subjects as rank A, B, or C in order of increasing probability of each cancer incidence. The current study aimed to validate the potential of AICS for cancer detection. AICS values were determined from the PFAA levels in subjects examined at Chiba Cancer Center Cohort, Mitsui Memorial Hospital, and Saihaku Hospital, and the cancer incidence was investigated. The sensitivities of rank C for cancer diagnosis within 1 year after AICS examination were 83.3% (10/12) for gastric, 50.0% (2/4) for lung, 46.2% (6/13) for colorectal, 50.0% (8/16) for prostate, 43.8% (7/16) for breast, and 50.0% (1/2) for uterine/ovarian cancer. The total cancer detection rate via AICS was 0.33% (34/10,245). The sensitivities during the maximum follow-up period of 6.2 years were 51.7% (15/29) for gastric, 18.2% (2/11) for lung, 28.6% (8/28) for colorectal, 36.4% (8/22) for prostate, 29.0% (9/31) for breast, and 33.3% (2/6) for uterine/ovarian cancers. In conclusion, AICS is a more useful method for evaluating the probability of cancer incidence than for predicting onset, suggesting that annual AICS should be recommended to detect any malignancy.