Background and Objective <p>Cefazolin is widely used for perioperative antibiotic prophylaxis and exhibits high protein binding. As only the unbound fraction exerts antimicrobial activity, understanding protein binding at the target site is essential. This study aimed to characterize the protein binding pattern of cefazolin in human knee and shoulder synovial fluid.</p> Methods <p>This prospective study included adult patients undergoing arthroscopic or open knee or shoulder surgery who received 2 g of intravenous cefazolin as part of routine perioperative antibiotic prophylaxis. Synovial fluid was aspirated immediately before arthroscope insertion or after joint capsule exposure. Total and unbound cefazolin were quantified using an ultra-high-performance liquid chromatography system coupled to mass spectrometry. Protein binding was calculated from paired total and unbound measurements. Concentration dependence was assessed by examining the relationship between the unbound fraction and the unbound concentration, with protein binding saturation defined by the presence of a plateau in this relationship. Statistical significance was defined as <i>p</i> &lt; .05.</p> Results <p>A total of 201 patients were included. Median total and unbound cefazolin concentrations in synovial fluid were 40.4 mg/L and 13.7 mg/L, respectively, corresponding to a median unbound fraction of 24.6%. The unbound fraction increased with rising unbound concentration (<i>r</i> = 0.38; <i>p</i> &lt; .001) without evidence of plateau formation within the observed range.</p> Conclusions <p>Cefazolin exhibits concentration-dependent, but not saturated, protein binding in human synovial fluid after single-dose perioperative antibiotic prophylaxis.</p>