In this work, we present a methodology for choosing the best experimental conditions for transferring protein Langmuir films onto solid substrates. As an example of applying the proposed methodology, we used monolayers of the protein bovine serum albumin, which is a very stable protein and is of great interest in the development of immunosensors. Langmuir-Blodgett (LB) films of this protein, on different solid substrates, were obtained and characterized as a function of pH, surface pressure, temperature, and contact angle. The compressibility modulus, the spreading entropy, and the fraction of desorbed protein sections were used as control parameters to find these conditions. A careful analysis of these parameters shows that there is a window on the values of these experimental parameters in which the LB films are best formed. Our methodology can be applied to other biomacromolecules to find the best conditions to form LB films from isotherm measurements.