Microbiology is a branch of biology which studies about small microorganisms. Its practicum aims to support the learning materials, give the experiences and the basic skills of the students in the field of microbiology, especially the practicum itself. Before doing the practicum, the students have to master the easily understood working procedure in doing the practicum which is the flowchart-based working procedure. Because the problems that acquired by the researcher in the field are based on the observations and interviews with the practitioners, the working procedures are still in form of the description (the explanations of the procedure are in form of words and points) which are not making practicum becomes easier. This development research aims to describe the validity of a flowchart-based microbiology practicum handbook (the handbook). The method used in this research is Tessmer Model, but only until the stage of one-to-one (individual test/legibility). The result shows that the flowchart-based microbiology practicum handbook achieves the criteria of very valid based on the three experts with the average percentage of 84,55%. Meanwhile, the individual test of this book achieves the good criteria with the average percentage of 70,31%, so this book for the students of biology education at the Faculty of Teacher and Training Education in Lambung Mangkurat University Banjarmasin is reasonable to be tested to the next stage so it can be used on the learning process on the microbiology practicum, and make the students become easier in doing biology practicum. Keywords: practicum handbook, microbiology, flowchart