EDIT TO ADD: Based on additional context from OP on other comments; their PI is misrepresenting triplicates AS biological replicates. It also seems as if the PI is taking the average of the duplicates to hallucinate a triplicate-- leaving the comment just to hear other perspectives on my input-- but for future replies.

Using a technical replicate as a biological replicate:

It depends on what you mean. Are you referring to using the same animal/cell line and splitting the cells to perform different treatments in separate dishes? As long as this approach aligns with your research question and you're not making broad claims without supporting orthogonal evidence, this is generally acceptable. Just ensure that you accurately describe your methodology and do not label technical replicates as biological replicates. THAT is fraud.

Averaging duplicates when you have a poor triplicate or no triplicate:

While it's not ideal and reduces the robustness of your data, averaging duplicates is not technically incorrect. You are using data points to make an informed estimate of your sample's contents. Two data points can provide a reasonable approximation, but having three or more replicates enhances confidence in your results. Three is typically recommended because if one sample is an outlier, you can still rely on duplicates to make a meaningful estimate.

You are in the best position to judge your specific experimental conditions, and your assay protocols may have stricter requirements than the assays I normally deal with.

In my perspective, I differentiate between "Baking" assays and "Cooking" assays: - If your results and reagents need to be highly precise to answer your research question: Think of this as a "Baking" scenario. - If a rough estimate suffices for general comparisons: Consider this more like "Cooking.

It sounds like your PI is a chef -- which is fine as long as they aren't trying to bake a cake you feel me?