Exhaled breath analysis is a non-invasive assessment tool that has shown promise in human diagnostics, and could greatly benefit research, management, and conservation of large whales. However, hormone assessment of whale respiratory vapor (blow) has been challenged by variable water content and unknown total volume of collected samples. To advance this technique, we investigated urea (a compound present in narrow range in circulation) as a normalizing factor to correct for blow sample concentration. Normalized progesterone, testosterone, and cortisol concentrations of 100 blow samples from 46 photo-identified North Atlantic right whales (Eubalaena glacialis) were more biologically relevant compared to absolute estimates, varying by sex, age class, or individual. Progesterone was elevated in adult females compared with other cohorts and highest in one independently confirmed pregnant female. For both sexes, testosterone was two-fold higher in reproductively mature whales but studied adult females showed the widest variation. Cortisol was present in relatively low concentrations in blow and demonstrated variation between individual whales, suggesting potential for studies of individual differences in adrenal activity. Incorporation of methodologies that normalize sample concentration are essential for blow hormone analysis of free-swimming whales, and measurement of urea could be used to optimize non-invasive physiological assessment of whales.