Two extrinsic fluorescent probes, 3-(dimethylamino)-8,9,10,11-tetrahydro-7H-cyclohepta[a]naphthalen-7-one (1) and 7-(dimethylamino)-2,3-dihydrophenanthren-4(1H)-one (2), are used to probe the unfolding of human serum albumin by sodium dodecyl sulfate (SDS). These probes respond separately to the polarity and H-bond-donating ability of their surroundings. Competitive binding experiments show that fluorophore 1 binds to site I (domain IIA) and 2 binds to site II (domain IIIA). The local acidity of 1 in site I is out of the sensing range of 1, whereas the local acidity of 2 in site II is calculated to be nearly zero on Catalans solvent acidity index. Both probes show that the first two equivalents of bound SDS result in a decrease in the local polarity of the binding sites. Each subsequent equivalent of SDS gives rise to a dramatic increase in polarity until HSA is saturated with seven molecules of SDS at the end of the specific binding domain. Compound 2 experiences an increase of acidity of 0.10 on Catalans solvent acidity index through seven equivalents of SDS, but the local acidity for 1 is still out of range. The increase in acidity experienced by 2 is greater than the increase in polarity. This result is consistent with greater exposure of the carbonyl group in 2, but not the bulk of 2, to the aqueous solvent in site II of the SDS-saturated HSA complex.