The aim of this study is to conceptualize website usability and develop a survey instrument to measure related concepts from the perspective of end users. We designed a three-stage methodology. First, concepts related to website usability were derived using content analysis technique. A total of 16 constructs measuring website usability were defined with their explanations and corresponding open codes. Second, a survey instrument was developed according to the defined open codes and the literature. The instrument was first validated using face validity, pilot testing (n = 30), and content validity (n = 40). Third, the survey instrument was validated using explanatory and confirmatory analyses. In the explanatory analysis, 785 questionnaires were collected from e-commerce website users to validate the factor structure of website usability. For confirmatory factor analysis, a new sample collected from 1086 users of e-commerce websites was used to confirm the measurement model. In addition, nomological validation was conducted by analyzing the effect of website usability concepts on three key factors: “continued intention to use”, “satisfaction”, and “brand loyalty”.