Aim The aim of this study was to develop a method to calculate the performance, and isolate error contributions occurring in a microbial surface sampling protocol. Methods and Results The experiments were conducted using a slip/peel tester to provide consistent pressure during the wipe collection. Fluorescence microscopy was used to count spores deposited on the coupon prior to sampling. The mean recovery efficiency (RE) as well as the efficiency of each step in the process was estimated by a recovery balance (RB), similar to a mass balance. Two studies were conducted in this work. In the first one, the recovery of spores from the solution (REsoln) was 57 center dot 7% (SD=8 center dot 0), while spores left on the glass surface after wiping (REb+c) was 2 center dot 8% (SD=2 center dot 4). The RE of spores adhered to the tube wall (REtube) and glass surface (REsurf) was 1 center dot 2% (SD=19 center dot 6) and 5 center dot 8% (SD=7 center dot 1), respectively. From the recovery balance, it was determined that 39 center dot 9% (SD=21 center dot 2) of spores were lost to the wipe (REwipe). The applicability of the RB method was demonstrated in a second study by examining the relative impact of parameters affecting spore collection including relative humidity, wipe material, wetting agent and nonporous surfaces. Conclusions The approach used in this study pointed out the need for a closer analysis of the complex interaction between spores and wipe material because a substantial percentage of spores were lost to the wipe. Significance and Impact of the Study The recovery balance, in association with independent controls, provides an account for error contribution and potential variability on each step of the sampling protocol. The approach is not meant to be a replacement for field or laboratory validation of wipe recoveries but promote the development of new collection methodologies and support protocol optimization in laboratory settings.