Usability Guidelines for Use Case Applications serves as an introduction to the general topic of usability, i.e., how user-friendly and efficient a THESEUS prototype is. In these guidelines, we emphasize the importance of usability testing, particularly during the development of a given THESEUS prototype. We discuss the many advantages of testing prototypes and products in terms of costs, product quality, and customer satisfaction. Usability testing can improve development productivity through more efficient design and fewer code revisions. It can help to eliminate over-design by emphasizing the functionality required to meet the needs of real users. Design problems can be detected earlier in the development process, saving both time and money. In these Guidelines we provide a brief overview of testing options, ranging from a cognitive walkthrough to interviews to eye tracking. Different techniques are used at different stages of a product's development. While many techniques can be applied, no single technique alone can ensure the usability of prototypes. Usability is a process with iterative steps, meaning the cycle is repeated but in a cumulative fashion, similar to software development. In order to test, a prototype must be available and we devote some time in the Guidelines to an overview of different tools and ways to build the necessary prototypes. We also describe some options such as paper prototyping, prototypes from Visio, PowerPoint, HTML, Flash and others, and working prototypes (Java, C++, etc.) before addressing the actual tests. Before any testing is conducted, the purpose of the test should be clarified. This will have considerable impact on the kind of testing to be done. A test plan should also be written before the start of the test which considers several different aspects including, for instance, the duration of the test, where it will take place, or who the experimenter will be. A pilot test is also recommended to avoid misunderstandings and other problems during the actual test. In this context, the Guidelines also discuss other important aspects such as budget, room set-up, time, and limitations of the experimenter and test subjects themselves. To provide an overview of some of the projects THESEUS is concerned with in the context of usability, we supply explicit recommendations that result in proposed scenarios for use cases in the Guidelines. The THESEUS program consists of six use cases: ALEXANDRIA, CONTENTUS, MEDICO, ORDO, PROCESSUS, and TEXO. In order to come up with the different testing scenarios, each of which has specific design and testing recommendations, we first extracted some substantial information from the different use cases in different user settings: we discerned between those who will use the system, where they will use the system, and what they will do with the system. After considering the results, we determined that the THESEUS program works with seven different scenarios. We provide a decision tree that leads to specific recommendations for designing and testing with prototypes for each of the different scenarios and user settings. General recommendations concerning various input methods, the design, and the testing itself have also been included in the Guidelines. Following that, we emphasize what we find important for the design and testing of each of the seven testing scenarios. We address, for instance, the appropriate input method (keyboard, mouse, speech, etc.), according to the type of test subject (e.g., administrator or mobile user), or also which prototype could be used for the usability test. We will also challenge the usability of traditional usability guidelines. Oftentimes, guideline descriptions and explanations are unsatisfactory, remaining vague and ambiguous in explanation The Guidelines close with an extensive list of recommended further information sources.