Most techniques for measuring tissue concentrations of drugs are invasive, time-consuming, and often require the removal of tissue or body fluids. Optical pharmacokinetics (OP) is a minimally invasive alternative giving an immediate result. Pulses of white light are directed at the tissue of interest using a fiber optic probe. Scattered light is detected by a second fiber immediately adjacent to the first in the same probe (separation 1.7 mm). Using the photosensitizer disulfonated aluminium phthalocyanine (AlS2Pc), OP measurements were made in phantoms and on the mouth, stomach, colon, skin, and liver of normal rats 1 and 24 h after intravenous AlS2Pc administration. AlS2Pc concentration was determined by calculating the area under the curve (AUC) in the spectral region around the peak drug absorption or measuring the height of the peak. Spectral baseline interpolation removed the need for pre-drug, control optical measurements. OP measurements correlated well with values from alkali chemical extraction (CE) of the corresponding tissues, (R2 0.87–0.97). OP measurements in the mouth also correlated with CE of less accessible internal organs (R2 0.77–0.88). In phantoms, the lowest detectable concentration was 0.1 μg/g. In vivo, results were limited by the lower accuracy in the CE measurements but were almost certainly comparable. An incidental finding was a 12–15 nm red shifted component in the spectra observed 1 h after drug administration, suggesting partitioning of the drug in different microenvironment compartments, which could prove to be of considerable interest in future studies. In conclusion, OP shows promise for real-time measurement of concentrations of drugs with suitable absorption peaks.