Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3–19.4 nmol/L in plasma) vs. refined wheat (0.05–2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma (p < 0.04). To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation.