Lysosomal accumulation of drugs with their specific physicochemical properties is of key importance to drug distribution in the body. Several attempts have been made to treat various human diseases by employing the accumulation of lysosomal drugs, and many methods to identify lysosomal accumulation of drugs have been proposed. Among those, the use of high-content screening has increased tremendously because of improved efficiency and accuracy as well as the development of automatic image acquisition and analytical techniques. Conventional methods to identify lysosomal accumulation of drugs by evaluating changes in the lysosomal area are unable to maximize the advantages of phenotypic high-content screening. Lysosomal distribution and the size of lysosomes are affected by lysosomal accumulating drugs. Therefore, we present image acquisition conditions and analytical methods to utilize lysosomal distribution and size as parameters for identifying lysosomal accumulating drugs. These two parameters will help to improve the reliability of the screening methods for identifying lysosomal accumulation of drugs by maximizing usage of information from image-based screening.