Highlights: • Diversified farming systems use agrobiodiversity in space and time to generate ecological processes replacing external inputs. • We discuss the main methodological challenges involved in assessing diversified farming systems. • We question the reference framework and indicators used and their spatial and temporal extents. • We recommend in-depth reflection about the reference systems or thresholds chosen for interpretation. • We recommend a complete overhaul of the methods used to assess diversified farming systems. Abstract: In response to criticism of current specialized and input-intensive agriculture, diversified farming systems (DFS) have increased in popularity. The most advanced forms of DFS include perennial components (e.g., perennial crops, trees), functionally different plants and livestock, and landscape heterogeneity. These characteristics make it more difficult to assess DFS in agricultural sciences, which have focused to date mainly on low-diversity systems. We provide an overview of the main methodological issues involved in assessing DFS, and their potential solutions. We identify five key methodological challenges for assessing DFS: i) combining conceptual frameworks from different disciplines to develop a holistic view of DFS multi-performance; ii) characterizing DFS structure from a functional perspective to define the relevant spatial extent to assess them; iii) considering DFS ontogeny i.e. the time required for DFS to establish, reach maturity and achieve a dynamic steady state when assessing their performance; iv) aggregating and mapping multiple performance indicators to integrate the range of products and services generated by DFS and to reveal potential synergies and antagonisms; v) defining and characterizing the reference systems used to compare DFS, as systems might differ in history, composition, management, soil-climate, and sociotechnical conditions. These challenges underline the need to overhaul assessments in agricultural sciences (e.g., agronomy, animal science, economics, ecology) to identify whether, and under what conditions, DFS are relevant and sustainable options for farmers. They also come with available options for researchers to proceed with this overhaul. Graphical abstract: https://ars.els-cdn.com/content/image/1-s2.0-S0308521X24000957-ga1.jpg