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Karyotype is a complete image of chromosomes which arranged according to the length, number, shape, and banded of an individual cells chromosomes. Generally, the karyotype is performed by taking the chromosome image at metaphase, cutting out, and identifying each chromosome image based on G-banded manually. This process consumes a lot of time, energy, and inconsistency result. Therefore, it is necessary to develop an application which helps the karyotyping process automatically or at least semi-automatically. In this experiment, we proposed a semi-automatic karyotyping process by utilizing image processing techniques of normal human karyotype. The development of semiautomatic application prototype includes three steps, they were preprocessed of chromosome image, feature extraction of chromosome image, and utilized tools to facilitate the users. The methods which used in preprocessed of chromosome image are gamma enhancement, bilateral filtering, adaptive thresholding, and flood-fill algorithms. The feature extraction of chromosome image is used to extract the morphological features, which will measure the chromosome based on length and pair of chromosomes using skeletonize method. Functionality tools join and cut for users to modify the results of segmentation chromosome image has been successfully created. Based on the analytical result, it can be concluded that the proposed method can provide a relatively good representation of the chromosomes to assist the further chromosome analysis process.