In cellular and molecular biology, fluorophores are employed to aid in tracking and quantifying molecules involved in cellular function. We previously developed a sensitive single-molecule quantification technique to count the number of proteins and the variation of the protein number over the population of individual subcellular organelles. However, environmental effects on the fluorescent intensity of fluorophores can make it difficult to accurately quantify proteins using these sensitive techniques. In this letter, we demonstrate the use of photobleaching to extract an accurate single-molecule calibration intensity distribution from the sample directly to avoid any differences in environment that may alter the count. Using this technique, we were able to show that goat antimouse IgG antibody labeled with Alexa Fluor 488, an environmentally insensitive fluorophore, exhibited an average fluorescence equivalent to 4.6 single fluorophores. SynaptopHluorin vesicles, which contain the environmentally sensitive green fluorescent protein, exhibited an average of 4.4 single green fluorescent proteins per vesicle.