Post-antibiotic effect (PAE) is the continued suppression of bacterial growth following a limited exposure to an antimicrobial agent. The presence of PAE needs consequential consideration in designing antibiotic dosage regimens. To understand the behavior of bacteria, PAE provides information on how long antibiotics are applied to the bacteria. Conventional methods of measuring PAE depend on population detection and have limitations for understanding the individual behavior of bacteria. To observe the PAE, we utilized an imaging technique with the use of microscopy. Here, we discuss the microscopic image analysis system we used to study the PAE at a single-colony level. The size and number of colonies of bacteria were measured prior to and following antibiotic removal. We could count a single colony, see the development of the settlement prior to and following exposure of antibiotics and track the colony by microscopy according to the incubation time and the image processed by our own image processing program. The PAE of antibiotics was quantified by comparing bacteria size and number based on their exposure time. In our study, we discovered that the longer exposure of antibiotics causes the bacteria to be suppressed—even after washing the antibiotics from the solution. This finding suggests that microscopic imaging detection provides a new method for understanding PAE. In addition, the behavior of the cell in response to drugs and chemicals and their removal can be examined with the use of single colony analysis.