Bovine serum albumin (BSA) was conjugated to three organic fluorescent probes, 5‐(4,6‐Dichloro‐s‐triazin‐2‐ylamino)fluorescein hydrochloride (DTAF), Rhodamine B isothiocyanate (RITC), and Lucifer yellow VS (LY). The protein sorption profile to one pHEMA‐based (etafilcon A) and three silicone hydrogel (SH) contact lens types (lotrafilcon B, balafilcon A and senofilcon A) was determined using confocal laser scanning microscopy. In addition, all lenses were incubated in dye solutions containing the fluorescent probe alone; and in a separate experiment BSA accumulation was quantified using radiolabeling. The different fluorescent conjugates showed similar sorption profiles for the pHEMA‐based lens, but marked differences for all SH lenses. Lotrafilcon B accumulated more protein on the surface as compared to the matrix, independent of the fluorescent probe used for conjugation. Protein sorption varied for senofilcon A, with DTAF‐BSA sorbing primarily to the surface region, while the other conjugates penetrated in equal amounts into the matrix. Balafilcon A exhibited smaller differences between conjugates, with LY‐BSA allowing the protein to fully penetrate the matrix, while the other conjugates showed minor surface adsorption. Sorption curves of unbound dyes were often similar compared to the conjugated results. BSA profiles to pHEMA‐based and silicone hydrogel lenses were highly dependent on the fluorescent probe used and none of the probes accurately reflected quantitative protein levels for the lens materials investigated. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010.